bluuebunny/crossref_metadata_2025 huggingface dataset:
Nasal IL-12p70 DNA Prevents and Treats Intestinal Allergic Diarrhea
Authors: Hino Ayako , Fukuyama Satoshi , Kataoka Kosuke , Kweon Mi-Na , Fujihashi Kohtaro , Kiyono Hiroshi
Published: April 2014

OVA-induced allergic diarrhea occurs as a consequence of over-expression of Th1 inhibitory IL-12p40 monomers and homodimers in the large intestine, establishing a dominant Th2-type environment. In this study, we demonstrate that intranasally administered murine IL-12p70 naked DNA expression plasmids resulted in the synthesis of corresponding cytokine in the large intestinal CD11c+ dendritic cells, leading to the inhibition of Ag-specific Th2-type response for the prevention of allergic diarrhea and the suppression of clinical symptoms including OVA-specific IgE Ab synthesis. The nasal IL-12p70 DNA treatment proved effective even after the establishment of allergic diarrhea. These results suggest that the mucosal administration of naked IL-12p70 DNA plasmid should be considered as a possible preventive and therapeutic treatment for Th2 cell-mediated food allergic diseases in the intestinal tract.
Initial High-Dose Nasal Allergen Exposure Prevents Allergic Sensitization to a Neoantigen
Authors: Riedl Marc A. , Landaw Elliot M. , Saxon Andrew , Diaz-Sanchez David
Published: April 2014

Primary allergic sensitization—IgE formation after Ag exposure—is fundamental in the development of allergic respiratory disease. With the rising prevalence of asthma and allergic rhinitis, improved understanding of the determining factors for allergic sensitization is needed. Human epidemiologic studies suggest high-dose allergen exposure may paradoxically protect against sensitization. Prospective human studies of allergen dose effect on primary allergic sensitization are lacking. We prospectively examined the effect of respiratory Ag dose exposure on the rate of primary allergic sensitization to a neoantigen, keyhole limpet hemocyanin, using a unique model of human nasal allergic sensitization. Atopic human subjects were exposed to 0.1-, 10-, 1,000-, or 100,000-μg doses of intranasal keyhole limpet hemocyanin in conjunction with adjuvant intranasal diesel exhaust particles. Ag-specific IgE, IgG, and IgG4 were measured in nasal lavage samples at the conclusion of the sensitization protocol. Allergic sensitization rates for the 0.1-, 10-, 1,000-, and 100,000-μg dose groups were 0, 100, 57, and 11%, respectively. All subjects produced Ag-specific IgG with the highest levels observed in the high-dose group. These results provide direct evidence that primary allergic sensitization may be prevented by initial high levels of respiratory Ag exposure through induction of a modified, nonallergic immune response. This Ag dose effect was capable of overcoming the well-established allergic adjuvant effects of diesel exhaust particle exposure. Whether this immune response represents durable allergic tolerance is not yet known. Studies investigating the molecular mechanisms of this non-IgE response may be useful in developing therapy to prevent allergic sensitization.
The Influence of Age on T Cell Generation and TCR Diversity
Authors: Naylor Keith , Li Guangjin , Vallejo Abbe N. , Lee Won-Woo , Koetz Kerstin , Bryl Ewa , Witkowski Jacek , Fulbright James , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

The ability to mount protective immune responses depends on the diversity of T cells. T cell diversity may be compromised by the declining thymic output of new T cells. The aging process imposes a threat to diversity, because thymic function deteriorates. In this study we have examined the relationship between thymic production, homeostatic T cell proliferation and TCR β-chain diversity in young (∼25 years), middle-aged (∼60 years), and elderly adults (∼75 years). TCR excision circles (TREC) as a marker of thymic output exponentially decreased by >95% between 25 and 60 years of age. The frequency of Ki67+ cycling CD4 T cells remained steady, and surprisingly, the diversity of the naive CD4 T cell repertoire was maintained at ∼2 × 107 different TCR β-chains. After the age of 70 years, TRECs only slightly declined, but homeostatic proliferation doubled. The diversity of the T cell pool drastically contracted to 200,000 TCR β-chains. Also, the phenotypic distinction between naive and memory CD4 T cells became fuzzy. The collapse in CD4 T cell diversity during the seventh and eighth decades indicates substantial T cell loss and implies that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment.
Leptin in Immunology
Authors: Matarese Giuseppe , Moschos Stergios , Mantzoros Christos S.
Published: April 2014

Leptin is an adipokine which conveys information on energy availability. In humans, leptin influences energy homeostasis and regulates neuroendocrine function primarily in states of energy deficiency. As a cytokine, leptin also affects thymic homeostasis and, similar to other proinflammatory cytokines, leptin promotes Th1 cell differentiation and cytokine production. We review herein recent advances on the role of leptin in the pathophysiology of immune responses.
Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity
Authors: Tangri Shabnam , Mothé Bianca R. , Eisenbraun Julie , Sidney John , Southwood Scott , Briggs Kristen , Zinckgraf John , Bilsel Pamuk , Newman Mark , Chesnut Robert , LiCalsi Cynthia , Sette Alessandro
Published: April 2014

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.
Clustering of T Cell Ligands on Artificial APC Membranes Influences T Cell Activation and Protein Kinase C θ Translocation to the T Cell Plasma Membrane
Authors: Giannoni Francesca , Barnett Joellen , Bi Kun , Samodal Rodrigo , Lanza Paola , Marchese Patrizia , Billetta Rosario , Vita Randi , Klein Mark R. , Prakken Berent , Kwok William W. , Sercarz Eli , Altman Amnon , Albani Salvatore
Published: April 2014

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase θ, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.
Impaired Cytolytic Activity in Calreticulin-Deficient CTLs
Authors: Sipione Simonetta , Ewen Catherine , Shostak Irene , Michalak Marek , Bleackley R. Chris
Published: April 2014

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt+/+ and crt−/− mice expressing a constitutively active form of calcineurin in the heart. Crt−/− CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt−/− cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt−/− CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.
Heat Shock Protein 60 Inhibits Th1-Mediated Hepatitis Model via Innate Regulation of Th1/Th2 Transcription Factors and Cytokines
Authors: Zanin-Zhorov Alexandra , Bruck Rafael , Tal Guy , Oren Shirly , Aeed Hussein , Hershkoviz Rami , Cohen Irun R. , Lider Ofer
Published: April 2014

Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-κB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-α and IFN-γ and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.
The Immunogenicity of Dendritic Cell-Based Vaccines Is Not Hampered by Doxorubicin and Melphalan Administration
Authors: Casati Anna , Zimmermann Valérie S. , Benigni Fabio , Bertilaccio Maria T. S. , Bellone Matteo , Mondino Anna
Published: April 2014

Immunization of cancer patients is most effective in tumor-free conditions or in the presence of minimal residual disease. In the attempt to develop new strategies able to control tumor recurrence while allowing the development of protective immunity, we have investigated the immunogenic potential of two distinct vaccine formulations when provided alone or upon single and repeated treatment with chemotherapeutics drugs. Vaccine-induced T cell responses were first investigated by tracing Ag-specific T cell responses in mice bearing detectable frequencies of Ag-specific TCR transgenic CD4 and CD8 T cells. These studies indicated that immunization with peptide-pulsed dendritic cells and soluble Ag plus adjuvant elicited a comparable expansion and differentiation of CD4 and CD8 effector cells in the peripheral lymphoid tissues when provided alone or shortly after Doxorubicin or Melphalan administration. We also analyzed the potency of the combined vaccination in transgenic adenocarcinoma mouse prostate mice, which develop spontaneous prostate cancer. Dendritic cell-based vaccination elicited potent tumor-specific cytotoxic responses in mice bearing prostate intraepithelial neoplasia both in the absence and in the presence of Doxorubicin. Together our results indicate that Doxorubicin- or Melphalan-based chemotherapy and Ag-specific vaccination can be combined for adjuvant treatments of cancer patients.
Mechanisms That Limit the In Vitro Proliferative Potential of Human CD8+ T Lymphocytes
Authors: Migliaccio Marco , Raj Kenneth , Menzel Olivier , Rufer Nathalie
Published: April 2014

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16INK4a is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16INK4a and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16INK4a expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16INK4a and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16INK4a limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.
Regulatory T Cells Suppress CD8+ T Cell Responses Induced by Direct Priming and Cross-Priming and Moderate Immunodominance Disparities
Authors: Haeryfar S. M. Mansour , DiPaolo Richard J. , Tscharke David C. , Bennink Jack R. , Yewdell Jonathan W.
Published: April 2014

Little is known regarding the participation of CD4+CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.
Opposing Roles for the Cyclin-Dependent Kinase Inhibitor p27kip1in the Control of CD4+ T Cell Proliferation and Effector Function
Authors: Rowell Emily A. , Walsh Matthew C. , Wells Andrew D.
Published: April 2014

Cell division drives T cell clonal expansion and differentiation, and is the result of concerted signaling from Ag, costimulatory, and growth factor receptors. How these mitogenic signals are coupled to the cell cycle machinery in primary T cells is not clear. We have focused on the role of p27kip1, a major cyclin-dependent kinase binding protein expressed by CD4+ T cells. Our studies using p27kip1 gene dosage demonstrate that early after activation, p27kip1 acts to promote, rather than inhibit, G1 to S phase progression within the first division cycle. However, throughout subsequent cell divisions p27kip1 behaves as a negative regulator, directly establishing the threshold amount of growth factor signaling required to support continued cell division. During this phase, signals from CD28 and IL-2R cooperate with the TCR to “tune” this threshold by inducing the degradation of p27kip1 protein, and we show that agents that block these pathways require elevated p27kip1 levels for their full antiproliferative activity. Finally, we show that p27kip1 opposes the development of CD4+ T cell effector function, and is required for the full development of anergy in response to a tolerizing stimulus. Our results suggest that p27kip1 plays a complex and important role in the regulation of cell division and effector function in primary CD4+ T cells.
B Cell Receptor (BCR) Cross-Talk: CD40 Engagement Enhances BCR-Induced ERK Activation
Authors: Mizuno Takuya , Rothstein Thomas L.
Published: April 2014

Bystander B cells may be initially stimulated through CD40, which enhances susceptibility to Fas-mediated apoptosis, before encountering Ag, which produces Fas resistance. A key issue in this process is to what extent CD40 cross-talk might affect subsequent BCR signaling. It has previously been shown that CD40 engagement bypasses or mitigates the need for Bruton’s tyrosine kinase in subsequent BCR signaling for NF-κB activation. However, the full extent of the effects of CD40 on BCR signaling has not been delineated. In the present study we evaluated the possibility that CD40-mediated cross-talk also affects another principal outcome of BCR signaling: MAPK activation. We found that prior stimulation of primary murine B cells with CD40L markedly enhanced the level of ERK and JNK (but not p38 MAPK) phosphorylation produced by subsequently added anti-Ig Ab, and much, but not all, of this enhancement was independent of PI3K and phospholipase C. CD40L treatment similarly enhanced BCR-induced MAPK kinase (MEK) phosphorylation, and MEK was required for enhancement of ERK. Although BCR-induced c-Raf phosphorylation was also enhanced by prior CD40L treatment, c-Raf was not required for MEK/ERK phosphorylation. These results identify a novel system of receptor cross-talk between CD40 and BCR and indicate that the effects of CD40 engagement on subsequent BCR stimulation spread beyond NF-κB to involve the MAPK pathway.
B and T Lymphocyte Attenuator Exhibits Structural and Expression Polymorphisms and Is Highly Induced in Anergic CD4+ T Cells
Authors: Hurchla Michelle A. , Sedy John R. , Gavrielli Maya , Drake Charles G. , Murphy Theresa L. , Murphy Kenneth M.
Published: April 2014

B and T lymphocyte attenuator (BTLA) was initially identified as expressed on Th1 cells and B cells, but recently reported to be expressed by macrophages, dendritic cells, and NK cells as well. To address this discrepancy we generated a panel of BTLA-specific mAbs and characterized BTLA expression under various activation conditions. We report the existence of three distinct BTLA alleles among 23 murine strains, differing both in Ig domain structure and cellular distribution of expression on lymphoid subsets. The BALB/c and MRL/lpr alleles differ at one amino acid residue, but C57BL/6 has nine additional differences and alters the predicted cysteine bonding pattern. The BALB/c BTLA allele is also expressed by B cells, T cells, and dendritic cells, but not macrophages or NK cells. However, C57BL/6 BTLA is expressed on CD11b+ macrophages and NK cells. Finally, in CD4+ T cells, BTLA is expressed most highly following Ag-specific induction of anergy in vivo, and unlike programmed death-1 and CTLA-4, not expressed by CD25+ regulatory T cells. These results clarify discrepancies regarding BTLA expression, suggest that structural and expression polymorphisms be considered when analyzing BTLA in various murine backgrounds, and indicate a possible role in anergic CD4+ T cells.
Role of the Programmed Death-1 Pathway in Regulation of Alloimmune Responses In Vivo
Authors: Sandner Sigrid E. , Clarkson Michael R. , Salama Alan D. , Sanchez-Fueyo Alberto , Domenig Christoph , Habicht Antje , Najafian Nader , Yagita Hideo , Azuma Miyuki , Turka Laurence A. , Sayegh Mohamed H.
Published: April 2014

Programmed death-1 (PD-1), an inhibitory receptor up-regulated on activated T cells, has been shown to play a critical immunoregulatory role in peripheral tolerance, but its role in alloimmune responses is poorly understood. Using a novel alloreactive TCR-transgenic model system, we examined the functions of this pathway in the regulation of alloreactive CD4+ T cell responses in vivo. PD-L1, but not PD-1 or PD-L2, blockade accelerated MHC class II-mismatched skin graft (bm12 (I-Abm12) into B6 (I-Ab)) rejection in a similar manner to CTLA-4 blockade. In an adoptive transfer model system using the recently described anti-bm12 (ABM) TCR-transgenic mice directly reactive to I-Abm12, PD-1 and PD-L1 blockade enhanced T cell proliferation early in the immune response. In contrast, at a later time point preceding accelerated allograft rejection, only PD-L1 blockade enhanced T cell proliferation. In addition, PD-L1 blockade enhanced alloreactive Th1 cell differentiation. Apoptosis of alloantigen-specific T cells was inhibited significantly by PD-L1 but not PD-1 blockade, indicating that PD-1 may not be the receptor for the apoptotic effect of the PD-L1-signaling pathway. Interestingly, the effect of PD-L1 blockade was dependent on the presence of CD4+CD25+ regulatory T cells in vivo. These data demonstrate a critical role for the PD-1 pathway, particularly PD-1/PD-L1 interactions, in the regulation of alloimmune responses in vivo.
ICAM-1 Contributes to but Is Not Essential for Tumor Antigen Cross-Priming and CD8+ T Cell-Mediated Tumor Rejection In Vivo
Authors: Blank Christian , Brown Ian , Kacha Aalok K. , Markiewicz Mary A. , Gajewski Thomas F.
Published: April 2014

ICAM-1 has been described to provide both adhesion and costimulatory functions during T cell activation. In the setting of antitumor immunity, ICAM-1/LFA-1 interactions could be important at the level of T cell priming by APCs in draining lymph nodes as well as for transendothelial migration and tumor cell recognition at the tumor site. To determine the contribution of ICAM-1 to tumor rejection in vivo, we performed adoptive transfer of 2C TCR-transgenic/RAG2−/− T cells into TCRα−/− vs ICAM−/−/TCRα−/− recipient animals. ICAM-1-deficient mice successfully rejected HTR.C tumors expressing Ld recognized by the 2C TCR, albeit with a kinetic delay. Inasmuch as HTR.C tumor cells themselves express ICAM-1, a second model was pursued using B16-F10 melanoma cells that lack ICAM-1 expression. These cells were transduced to express the SIYRYYGL peptide recognized by the 2C TCR in the context of Kb, which is cross-presented by APCs in H-2b mice in vivo. These tumors also grew more slowly but were eventually rejected by the majority of ICAM-1−/−/TCRα−/− recipients. Delayed rejection in ICAM-1−/− mice was associated with diminished T cell priming as assessed by ELISPOT. In contrast, T cell penetration into the tumor was comparable in wild-type and ICAM-1−/− hosts, and adoptively transferred primed effector 2C cells rejected normally in ICAM-1−/− recipients. Our results suggest that ICAM-1 contributes to but is not absolutely required for CD8+ T cell-mediated tumor rejection in vivo and dominantly acts at the level of priming rather than the effector phase of the antitumor immune response.
Cross-Priming of T Cell Responses by Synthetic Microspheres Carrying a CD8+ T Cell Epitope Requires an Adjuvant Signal
Authors: Boisgérault Florence , Rueda Paloma , Sun Cheng Ming , Hervas-Stubbs Sandra , Rojas Marie , Leclerc Claude
Published: April 2014

Controlling the cross-presentation of exogenous Ags to CD8+ T cells represents a major step for designing new vaccination strategies. Whereas several recombinant pseudo-viral particles have been used as delivery systems for triggering potent CTL responses to heterologous exogenous Ags, the adjuvant properties of virus-like particles (VLPs) themselves were little questioned. Here, we analyzed the contribution of the porcine parvovirus (PPV)-VLPs to the induction of protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway, were chosen for delivering the immunodominant OVA257–264 CD8+ T cell epitope (B-OVAp). This delivery system fulfills the requirements in terms of cross-presentation, but fails to induce cross-priming of specific CD8+ T cells. Coinjection of PPV-VLPs with B-OVAp results in the priming of potent CTL responses and type 1-biased immunity in a CD4- and CD40-independent manner, as efficiently as the recombinant PPV-VLPs carrying the same epitope (PPV-OVAp). Furthermore, vaccination with PPV-VLPs and B-OVAp was fully efficient to protect mice against the development of OVA-bearing melanoma. These findings indicate that PPV-VLPs act not only as a delivery system but also as a strong adjuvant when independently provided with exogenous Ag. Thus, dissociation between delivery system and adjuvant would provide a more flexible and reliable system to induce potent and protective CTL.
Genes “Waiting” for Recruitment by the Adaptive Immune System: The Insights from Amphioxus
Authors: Yu Cuiling , Dong Meiling , Wu Xiaokun , Li Shengguo , Huang Shengfeng , Su Jing , Wei Jianwen , Shen Yang , Mou Chunyan , Xie Xiaojin , Lin Jianghai , Yuan Shaochun , Yu Xuesong , Yu Yanhong , Du Jingchun , Zhang Shicui , Peng Xuanxian , Xiang Mengqing , Xu Anlong
Published: April 2014

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.
Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition
Authors: Mielenz Dirk , Vettermann Christian , Hampel Martin , Lang Christiane , Avramidou Athanasia , Karas Michael , Jäck Hans-Martin
Published: April 2014

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits α-1 and β-2, vimentin, γ-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.
The IL-27 Receptor Chain WSX-1 Differentially Regulates Antibacterial Immunity and Survival during Experimental Tuberculosis
Authors: Hölscher Christoph , Hölscher Alexandra , Rückerl Dominik , Yoshimoto Takayuki , Yoshida Hiroki , Mak Tak , Saris Christiaan , Ehlers Stefan
Published: April 2014

IL-12 is a potent inducer of IFN-γ production and promotes a protective cell-mediated immune response after Mycobacterium tuberculosis infection. Recently, the IL-12-related cytokine IL-27 was discovered, and WSX-1 was identified as one component of the IL-27R complex. To determine the functional significance of IL-27/WSX-1 during tuberculosis, we analyzed the course of infection and the immune response in WSX-1-KO mice after aerosol infection with M. tuberculosis. In the absence of WSX-1, an increased production of the proinflammatory cytokines TNF and IL-12p40 resulted in elevated CD4+ T cell activation and IFN-γ production, which enhanced macrophage effector functions and reduced bacterial loads. This is the first occasion of a selectively gene-deficient mouse strain showing higher levels of protective immunity against M. tuberculosis infection than wild-type mice. However, a concomitantly increased chronic inflammatory response also accelerated death of infected WSX-1-KO mice. In vitro, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation. In conclusion, IL-27 both prevents optimal antimycobacterial protection and limits the pathological sequelae of chronic inflammation.
Sphingosine Kinase 1 (SK1) Is Recruited to Nascent Phagosomes in Human Macrophages: Inhibition of SK1 Translocation by Mycobacterium tuberculosis
Authors: Thompson Christopher R. , Iyer Shankar S. , Melrose Natalie , VanOosten Rebecca , Johnson Korey , Pitson Stuart M. , Obeid Lina M. , Kusner David J.
Published: April 2014

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.
Mechanisms of the Natural Reactivity of Lymphocytes from Noninfected Individuals to Membrane-AssociatedLeishmania infantumAntigens
Authors: Sassi Atfa , Larguèche-Darwaz Beya , Collette Alexis , Six Adrien , Laouini Dhafer , Cazenave Pierre André , Dellagi Koussay
Published: April 2014

Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRαβ+, CD45RO+, and CD45RA+ and secrete IFN-γ and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-α, but have no effect on IL-1α or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable β segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host’s natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.
Intracellular IL-1 Receptor Antagonist Type 1 Inhibits IL-1-Induced Cytokine Production in Keratinocytes through Binding to the Third Component of the COP9 Signalosome
Authors: Banda Nirmal K. , Guthridge Carla , Sheppard Devon , Cairns Kelly S. , Muggli Michele , Bech-Otschir Dawadschargal , Dubiel Wolfgang , Arend William P.
Published: April 2014

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IκB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1α-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1α-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.
The Nuclear IκB Protein IκBNS Selectively Inhibits Lipopolysaccharide-Induced IL-6 Production in Macrophages of the Colonic Lamina Propria
Authors: Hirotani Tomonori , Lee Pui Y. , Kuwata Hirotaka , Yamamoto Masahiro , Matsumoto Makoto , Kawase Ichiro , Akira Shizuo , Takeda Kiyoshi
Published: April 2014

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMφ). CLPMφ of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMφ gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMφ. These genes included nuclear IκB proteins such as Bcl-3 and IκBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-α production, we analyzed the role of IκBNS in macrophages. Lentiviral introduction of IκBNS resulted in impaired LPS-induced IL-6 production, but not TNF-α production in the murine macrophage cell line RAW264.7. IκBNS expression led to constitutive and intense DNA binding of NF-κB p50/p50 homodimers. IκBNS was recruited to the IL-6 promoter, but not to the TNF-α promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IκBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-α. Thus, IκBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IκB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.
Manifestations of Inflammatory Arthritis Are Critically Dependent on LFA-1
Authors: Watts Gerald M. , Beurskens Frank J. M. , Martin-Padura Ines , Ballantyne Christie M. , Klickstein Lloyd B. , Brenner Michael B. , Lee David M.
Published: April 2014

Leukocyte infiltration of synovial fluid and tissues is the hallmark of inflammatory arthritis. Selectins and β2 integrins have been implicated in the multistep process of leukocyte adhesion to vascular endothelium. However, previous work has revealed disparate requirements for leukocyte recruitments to specific anatomic locales. Moreover, the mechanisms regulating recruitment of leukocytes to the joint in inflammatory arthritis models are not fully understood. We hypothesized that β2 integrins, expressed on leukocytes, might play a pathogenic role in synovial inflammation. Using mice deficient in all β2 integrins (CD18 null mice), we demonstrate that expression of these heterodimeric adhesion molecules is critical for arthritis induction in the K/B × N serum transfer model. Using null-allele mice and blocking mAbs, we demonstrate specifically that CD11a/CD18 (LFA-1) is absolutely required for the development of arthritis in this model. Blocking mAbs further revealed an ongoing requirement for LFA-1 I-domain adhesive function in disease perpetuation. These findings suggest that the LFA-1 I-domain forms an attractive target for treatment of human inflammatory arthritis.
B Cell Response to Surface IgM Cross-Linking Identifies Different Prognostic Groups of B-Chronic Lymphocytic Leukemia Patients
Authors: Nédellec Steven , Renaudineau Yves , Bordron Anne , Berthou Christian , Porakishvili Nina , Lydyard Peter M. , Pers Jacques-Olivier , Youinou Pierre
Published: April 2014

On the basis of responses to surface IgM (sIgM) cross-linking, B cells from 41 patients with B-chronic lymphocytic leukemia were categorized as 15 nonresponders (group I) and 26 responders (group II). The latter cases were subclassified as those seven where proliferation was induced (subgroup IIa) and the remaining 19 in whom apoptosis occurred (subgroup IIa). Signal disruption in group I was confirmed by the absence of Ca2+ mobilization. Activation of PI3K was constitutive in subgroup IIa, but not in subgroup IIb, and that of Akt induced by anti-μ in subgroup IIa, but not in subgroup IIb. Among the MAPK, ERK was more highly activated relative to p38 in subgroup IIa, whereas activation of p38 predominated over that of ERK in subgroup IIb. For subgroup IIb cells, based on tyrosine phosphorylation and translocation into lipid rafts, sIgM signaling was shown to be enhanced by Zap70. The different consequences of signaling through sIgM were associated with biological prognosis indicators. These included high levels of CD38, lack of mutations in the IgVH chain genes, preferential usage of full-length CD79b, and severe clinical stage. Thus, modification of sIgM-induced signaling could be a therapeutic approach.
CD1a and CD1c Activate Intrathyroidal T Cells during Graves’ Disease and Hashimoto’s Thyroiditis
Authors: Roura-Mir Carme , Catálfamo Marta , Cheng Tan-Yun , Marqusee Ellen , Besra Gurdyal S. , Jaraquemada Dolores , Moody D. Branch
Published: April 2014

Molecular studies have shown that CD1 proteins present self and foreign lipid Ags to T cells, but the possible roles of CD1 in human autoimmune diseases in vivo are not known, especially for the group 1 CD1 isoforms (CD1a, CD1b, and CD1c). To investigate the hypothesis that CD1-restricted T cells might be activated and home to target tissues involved in Hashimoto’s thyroiditis and Graves’ disease, we performed ex vivo analysis of lymphocytes from peripheral blood and autoinflammatory lesions of thyroid tissue. Immunofluorescence analysis identified two types of CD1-expressing APCs in inflamed thyroid tissues. CD1a, CD1b, and CD1c were expressed on CD83+ dendritic cells, and CD1c was expressed on an abundant population of CD20+IgD+CD23−CD38− B cells that selectively localized to the mantle zone of lymphoid follicles within the thyroid gland. CD1c-restricted, glycolipid-specific T cells could not be detected in the peripheral blood, but were present in polyclonal lymphocyte populations isolated from affected thyroid glands. In addition, polyclonal thyroid-derived lymphocytes and short-term T cell lines were found to recognize and lyse targets in a CD1a- or CD1c-dependent manner. The targeting of CD1-restricted T cells and large numbers of CD1-expressing APCs to the thyroid gland during the early stages of autoimmune thyroiditis suggests a possible effector function of CD1-restricted T cells in tissue destruction and point to a new model of organ-specific autoimmune disease involving lipid Ag presentation.
Mycobacterium tuberculosisHeat Shock Fusion Protein Enhances Class I MHC Cross-Processing and -Presentation by B Lymphocytes
Authors: Tobian Aaron A. R. , Harding Clifford V. , Canaday David H.
Published: April 2014

Exogenous heat shock protein (HSP):peptide complexes are processed for cross-presentation of HSP-chaperoned peptides on class I MHC (MHC-I) molecules. Fusion proteins containing HSP and Ag sequences facilitate MHC-I cross-presentation of linked antigenic epitopes. Processing of HSP-associated Ag has been attributed to dendritic cells and macrophages. We now provide the first evidence to show processing of HSP-associated Ag for MHC-I cross-presentation by B lymphocytes. Fusion of OVA sequence (rOVA, containing OVA230–359 sequence) to Mycobacterium tuberculosis HSP70 greatly enhanced rOVA processing and MHC-I cross-presentation of OVA257–264:Kb complexes by B cells. Enhanced processing was dependent on linkage of rOVA sequence to HSP70. M. tuberculosis HSP70-OVA fusion protein enhanced cross-processing by a CD91-dependent process that was independent of TLR4 and MyD88. The enhancement occurred through a post-Golgi, proteasome-independent mechanism. These results indicate that HSPs enhance delivery and cross-processing of HSP-linked Ag by B cells, which could provide a novel contribution to the generation of CD8+ T cell responses. HSP fusion proteins have potential advantages for use in vaccines to enhance priming of CD8+ T cell responses.
TGF-β1 Attenuates the Acquisition and Expression of Effector Function by Tumor Antigen-Specific Human Memory CD8 T Cells
Authors: Ahmadzadeh Mojgan , Rosenberg Steven A.
Published: April 2014

TGF-β1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-β1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-β1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-β1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-β1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-β1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-β1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients.
Cellular FLIP (Long Form) Regulates CD8+ T Cell Activation through Caspase-8-Dependent NF-κB Activation
Authors: Dohrman Austin , Kataoka Takao , Cuenin Solange , Russell Jennifer Q. , Tschopp Jurg , Budd Ralph C.
Published: April 2014

Cellular FLIP long form (c-FLIPL) was originally identified as an inhibitor of Fas (CD95/Apo-1). Subsequently, additional functions of c-FLIPL were identified through its association with receptor-interacting protein (RIP)1 and TNFR-associated factor 2 to activate NF-κB, as well as by its association with and activation of caspase-8. T cells from c-FLIPL-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIPL was involved. We have further explored the effect of c-FLIPL on CD8+ effector T cell function and its mechanism of action. c-FLIPL-Tg CD8+ T cells have increased proliferation and IL-2 responsiveness to cognate Ags as well as to low-affinity Ag variants, due to increased CD25 expression. They also have a T cytotoxic 2 cytokine phenotype. c-FLIPL-Tg CD8+ T cells manifest greater caspase activity and NF-κB activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIPL itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43FLIP form. p43FLIP more efficiently recruits RIP1 than full-length c-FLIPL to activate NF-κB. c-FLIPL and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8+ T cells. Thus, one mechanism by which c-FLIPL influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIPL to allow RIP1 recruitment and NF-κB activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.