bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-Cell Repertoire Analysis Demonstrates that Clonal Expansion Is a Prominent Feature of the B Cell Response in Multiple Sclerosis Cerebrospinal Fluid
Authors: Owens Gregory P. , Ritchie Alanna M. , Burgoon Mark P. , Williamson R. Anthony , Corboy John R. , Gilden Donald H.
Published: April 2014

Single-cell RT-PCR was used to sample CD19+ B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominately polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for ∼70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.
Diversity in Intrinsic Strengths of the Human Complement System: Serum C4 Protein Concentrations Correlate withC4Gene Size and Polygenic Variations, Hemolytic Activities, and Body Mass Index
Authors: Yang Yan , Chung Erwin K. , Zhou Bi , Blanchong Carol A. , Yu C. Yung , Füst George , Kovács Margit , Vatay Ágnes , Szalai Csaba , Karádi István , Varga Lilian
Published: April 2014

Among the genes and proteins of the human immune system, complement component C4 is extraordinary in its frequent germline variation in the size and number of genes. Definitive genotypic and phenotypic analyses were performed on a central European population to determine the C4 polygenic and gene size variations and their relationships with serum C4A and C4B protein concentrations and hemolytic activities. In a study population of 128 healthy subjects, the number of C4 genes present in a diploid genome varied between two to five, and 77.4% of the C4 genes belonged to the long form that contains the endogenous retrovirus HERV-K(C4). Intriguingly, higher C4 serum protein levels and higher C4 hemolytic activities were often detected in subjects with short C4 genes than those with long genes only, suggesting a negative epistatic effect of HERV-K(C4) on the expression of C4 proteins. Also, the body mass index appeared to affect the C4 serum levels, particularly in the individuals with medium or high C4 gene dosages, a phenomenon that was dissimilar in several aspects from the established correlation between body mass index and serum C3. As expected, there were strong, positive correlations between total C4 gene dosage and serum C4 protein concentrations, and between serum C4 protein concentrations and C4 hemolytic activities. There were also good correlations between the number of long genes with serum levels of C4A, and the number of short genes with serum levels of C4B. Thus, the polygenic and gene size variations of C4A and C4B contribute to the quantitative traits of C4 with a wide range of serum protein levels and hemolytic activities, and consequently the power of the innate defense system.
Identification of Novel IL-4/Stat6-Regulated Genes in T Lymphocytes
Authors: Chen Zhi , Lund Riikka , Aittokallio Tero , Kosonen Minna , Nevalainen Olli , Lahesmaa Riitta
Published: April 2014

IL-4, primarily produced by T cells, mast cells, and basophiles, is a cytokine which has pleiotropic effects on the immune system. IL-4 induces T cells to differentiate to Th2 cells and activated B lymphocytes to proliferate and to synthesize IgE and IgG1. IL-4 is particularly important for the development and perpetuation of asthma and allergy. Stat6 is the protein activated by signal transduction through the IL-4R, and studies with knockout mice demonstrate that Stat6 is critical for a number of IL-4-mediated functions including Th2 development and production of IgE. In the present study, novel IL-4- and Stat6-regulated genes were discovered by using Stat6−/− mice and Affymetrix oligonucleotide arrays. Genes regulated by IL-4 were identified by comparing the gene expression profile of the wild-type T cells induced to polarize to the Th2 direction (CD3/CD28 activation + IL-4) to gene expression profile of the cells induced to proliferate (CD3/CD28 activation alone). Stat6-regulated genes were identified by comparing the cells isolated from the wild-type and Stat6−/− mice; in this experiment the cells were induced to differentiate to the Th2 direction (CD3/CD28 activation + IL-4). Our study demonstrates that a number a novel genes are regulated by IL-4 through Stat6-dependent and -independent pathways. Moreover, elucidation of kinetics of gene expression at early stages of cell differentiation reveals several genes regulated rapidly during the process, suggesting their importance for the differentiation process.
Protein Kinase A Regulatory Subunit Type IIβ Directly Interacts with and Suppresses CREB Transcriptional Activity in Activated T Cells
Authors: Elliott Michael R. , Tolnay Mate , Tsokos George C. , Kammer Gary M.
Published: April 2014

Levels of the type IIβ regulatory subunit (RIIβ) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RIIβ in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RIIβ can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RIIβ-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RIIβ resulted in nuclear localization of a portion of the ectopically expressed RIIβ. In vitro and in vivo analyses revealed a novel, specific interaction between RIIβ and CREB that mapped to the N-terminal 135 aa of RIIβ. In functional studies, RIIβ inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RIIβ completely abrogated inhibition. Additionally, RIIβ suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RIIβ can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RIIβ in systemic lupus erythematosus T cells.
CD11b+ Peyer’s Patch Dendritic Cells Secrete IL-6 and Induce IgA Secretion from Naive B Cells
Authors: Sato Ayuko , Hashiguchi Masaaki , Toda Etsuko , Iwasaki Akiko , Hachimura Satoshi , Kaminogawa Shuichi
Published: April 2014

Peyer’s patch (PP) dendritic cells (DCs) have been shown to exhibit a distinct capacity to induce cytokine secretion from CD4+ T cells compared with DCs in other lymphoid organs such as the spleen (SP). In this study, we investigated whether PP DCs are functionally different from DCs in the SP in their ability to induce Ab production from B cells. Compared with SP DCs, freshly isolated PP DCs induced higher levels of IgA secretion from naive B cells in DC-T cell-B cell coculture system in vitro. The IgA production induced by PP DCs was attenuated by neutralization of IL-6. In addition, the induction of IgA secretion by SP DCs, but not PP DCs, was further enhanced by the addition of exogenous IL-6. Finally, we demonstrated that only PP CD11b+ DC subset secreted higher levels of IL-6 compared with other DC subsets in the PP and all SP DC populations, and that PP CD11b+ DC induced naive B cells to produce higher levels of IgA compared with SP CD11b+ DC. These results suggest a unique role of PP CD11b+ DCs in enhancing IgA production from B cells via secretion of IL-6.
Cysteine Protease B of Leishmania mexicana Inhibits Host Th1 Responses and Protective Immunity
Authors: Buxbaum Laurence U. , Denise Hubert , Coombs Graham H. , Alexander James , Mottram Jeremy C. , Scott Phillip
Published: April 2014

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Δcpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Δcpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Δcpb-infected IL-12p40−/− and STAT4−/− mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-γ response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.
Impacts of Avidity and Specificity on the Antiviral Efficiency of HIV-1-Specific CTL
Authors: Yang Otto O. , Sarkis Phuong T. Nguyen , Trocha Alicja , Kalams Spyros A. , Johnson R. Paul , Walker Bruce D.
Published: April 2014

Although CD8+ CTLs are presumed to be an important mediator of protective immunity in HIV-1 infection, the factors that determine CTL antiviral efficiency are poorly understood. Two factors that have been proposed to influence CTL antiviral function are antigenic avidity and epitope specificity. In this study we evaluate these by examining the activity of HIV-1-specific CTL against acutely infected cells. The ability of CTL to kill infected cells is variable and depends more on epitope specificity than functional avidity within the range for the tested clones (50% of maximal killing, 50 pg/ml to 100 ng/ml); killing efficiency is similar for different clones recognizing the same epitope, despite their variation in avidity. When CTL clones are tested for their ability to suppress viral replication, similar results are observed. Inhibition is more dependent on epitope specificity than functional avidity among the tested clones (50% of maximal killing, 20 pg/ml to 20 ng/ml). Thus, CTL specificity can be an overriding factor in the ability of CTL to interact with HIV-1-infected cells, indicating that factors determining the process of epitope presentation on infected cells have a key influence on CTL efficiency. These results suggest that CTL specificity may have a pivotal role in the immunopathogenesis of infection, and that simple quantitative measures of CTL may be insufficient indicators of the CTL response to HIV-1.
Highly Biased Type 1 Immune Responses in Mice Deficient in LFA-1 inListeria monocytogenesInfection Are Caused by Elevated IL-12 Production by Granulocytes
Authors: Emoto Masashi , Miyamoto Mamiko , Emoto Yoshiko , Yoshizawa Izumi , Brinkmann Volker , van Rooijen Nico , Kaufmann Stefan H. E.
Published: April 2014

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-γ producers in the liver and spleen of LFA-1−/− mice were markedly increased compared with heterozygous littermates and Vα14+NKT cell-deficient mice, and NK cells were major IFN-γ producers. Numbers of IL-12 producers were also markedly elevated in LFA-1−/− mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-γ production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-γ-secreting NK cells in the liver of LFA-1−/− mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1−/− mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1−/− mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-γ secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.
Burn Injury Promotes Antigen-Driven Th2-Type Responses In Vivo
Authors: Guo Zhijun , Kavanagh Eamon , Zang Yan , Dolan Sinead M. , Kriynovich Sara J. , Mannick John A. , Lederer James A.
Published: April 2014

Severe injury induces detrimental changes in immune function, often leaving the host highly susceptible to developing life-threatening opportunistic infections. Advances in our understanding of how injury influences host immune responses suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2-type immune responses. We report in this study, using a TCR transgenic CD4+ T cell adoptive transfer approach, that injury skews T cell responses toward increased Th2-type reactivity in vivo without substantially limiting Ag-driven CD4+ T cell expansion. The increased Th2-type response did not occur unless injured mice were immunized with specific Ag, suggesting that the phenotypic switch is Ag dependent. These findings establish that severe injury induces fundamental changes in the induction of Ag-specific CD4+ Th cell responses favoring the development of Th2-type immune reactivity in vivo.
Th1 Cytokines Regulate Adenosine Receptors and Their Downstream Signaling Elements in Human Microvascular Endothelial Cells
Authors: Khoa Nguyen D. , Montesinos M. Carmen , Williams Adrienne J. , Kelly Maureen , Cronstein Bruce N.
Published: April 2014

We and others have shown that adenosine, acting at its receptors, is a potent modulator of inflammation and angiogenesis. To better understand the regulation of adenosine receptors during these processes we studied the effects of IL-1, TNF-α, and IFN-γ on expression and function of adenosine receptors and select members of their coupling G proteins in human dermal microvascular endothelial cells (HMVEC). HMVEC expressed message and protein for A2A and A2B, but not A1 or A3 receptors. IL-1 and TNF-α treatment increased message and protein expression of A2A and A2B receptor. IFN-γ treatment also increased the expression of A2B receptors, but decreased expression of A2A receptors. Resting HMVEC and IFN-γ-treated cells showed minimal cAMP response to the selective A2A receptor agonist 2-[2-(4-chlorophenyl)ethoxy]adenosine (MRE0094). In contrast, MRE0094 stimulated a dose-dependent increase in cAMP levels in TNF-α-treated cells that was almost completely blocked by the A2A receptor antagonist ZM-241385 (4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol). The nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine increased cAMP levels in both TNF-α- and IFN-γ-treated cells, but not control cells, and its effect was only partially reversed by ZM-241385 in TNF-α-treated cells and not affected in IFN-γ-treated cells. HMVEC expressed a higher level of G protein β1 isoform than β4 isoform. Although none of the cytokines tested affected Gβ1 expression, both IL-1 and TNF-α significantly up-regulated Gβ4 expression. These findings indicate that inflammatory cytokines modulate adenosine receptor expression and function on HMVECs and suggest that the interaction between proinflammatory cytokines and adenosine receptors may affect therapeutic responses to anti-inflammatory drugs that act via adenosine-dependent mechanisms.
MHC Class I Gene Conversion Mutations Alter the CD8 T Cell Repertoire
Authors: Block Matthew S. , Hansen Michael J. , Van Keulen Virginia P. , Pease Larry R.
Published: April 2014

MHC class I molecules are highly polymorphic within populations. This diversity is thought to be the result of selective maintenance of new class I alleles formed by gene conversion. It has been proposed that rare alleles are maintained by their ability to confer resistance to common pathogens. Investigation has focused on differences in the presentation of foreign Ags by class I alleles, but the majority of peptides presented by class I molecules are self peptides used in shaping the naive T cell repertoire. We propose that the key substrate for the natural selection of class I gene conversion variants is the diversity in immune potential formed by new alleles. We show that T cells compete with each other for niches in the thymus and spleen during development, and that competition between different clones is dramatically affected by class I mutations. We also show that peripheral naive T cells proliferate preferentially in the presence of the class I variant that directed T cell development. The data argue that class I gene conversion mutations dramatically affect both the development and the maintenance of the naive CD8 T cell repertoire.
Expression of CD1d Under the Control of a MHC Class Ia Promoter Skews the Development of NKT Cells, But Not CD8+ T Cells
Authors: Xu Honglin , Chun Taehoon , Colmone Angela , Nguyen Hanh , Wang Chyung-Ru
Published: April 2014

Although CD1d and MHC class Ia share similar overall structure, they have distinct levels and patterns of surface expression. While the expression of CD1d1 is known to be essential for the development of NKT cells, the contribution of CD1d1 to the development of CD8+ T cells appears to be inconsequential. To investigate whether CD1d tissue distribution and expression levels confer differential capacity in selecting these two T cell subsets, we analyzed CD8 and NKT cell compartments in Kb-CD1d-transgenic mice that lack endogenous MHC class Ia and CD1d, respectively. We found that MHC class Ia-like expression pattern and tissue distribution are not sufficient for CD1d to rescue the development of CD8+ T cells, suggesting that unique structural features of CD1d preclude its active participation in selection of CD8+ T cells. Conversely, cell type-specific CD1d surface density is important for the selection of NKT cells, as the NKT cell compartment was only partially rescued by the Kb-CD1d transgene. We have previously demonstrated that increased CD1d expression on dendritic cells enhanced negative selection of NKT cells. In this study, we show that cell type-specific expression levels of CD1d establish a narrow window between positive and negative selection, suggesting that the distinct CD1d expression pattern may be selected evolutionarily to ensure optimal output of NKT cells.
Exposure of Human Primary Colon Carcinoma Cells to Anti-Fas Interactions Influences the Emergence of Pre-existing Fas-Resistant Metastatic Subpopulations
Authors: Liu Kebin , McDuffie Elwood , Abrams Scott I.
Published: April 2014

Fas, an important death receptor-mediated signaling pathway, has been shown to be down-regulated during human colon tumorigenesis; however, how alterations in Fas expression influence the metastatic process remains unresolved. In mouse models, loss of Fas function was found to be both necessary and sufficient for tumor progression. In this study, we investigated the link between functional Fas status and malignant phenotype using a matched pair of naturally occurring primary (Fas-sensitive) and metastatic (Fas-resistant) human colon carcinoma cell lines in both in vitro and in vivo (xenograft) settings. Metastatic sublines were produced in vitro from the primary tumor cell line by functional elimination of Fas-responsive cells. Conversely, sublines derived from the primary tumor in vivo at distal metastatic sites were Fas-resistant. In contrast, simply disrupting the Fas pathway by molecular-based strategies in the Fas-sensitive primary tumor failed to achieve the same metastatic outcome. Interestingly, both in vitro- and in vivo-produced sublines resembled the naturally occurring metastatic population, based on functional and morphologic studies and genome-scale gene expression profiling. Overall, using this human colon carcinoma model, we: 1) showed that loss of Fas function was linked to, but alone was insufficient for, acquisition of a detectable metastatic phenotype; 2) demonstrated that metastatic subpopulations pre-existed within the heterogeneous primary tumor, and that anti-Fas interactions served as a selective pressure for their outgrowth; and 3) identified a large set of differentially expressed genes distinguishing the primary from metastatic malignant phenotypes. Thus, Fas-based interactions may represent a novel mechanism for the biologic or immunologic selection of certain types of Fas-resistant neoplastic clones with enhanced metastatic ability.
Receptor Glycosylation Regulates Ly-49 Binding to MHC Class I
Authors: Mason Llewellyn H. , Willette-Brown Jami , Anderson Stephen K. , Alvord W. Gregory , Klabansky Richard L. , Young Howard A. , Ortaldo John R.
Published: April 2014

Murine NK cells express the Ly-49 family of class I MHC-binding receptors that control their ability to lyse tumor or virally infected host target cells. X-ray crystallography studies have identified two predominant contact sites (sites 1 and 2) that are involved in the binding of the inhibitory receptor, Ly-49A, to H-2Dd. Ly-49G2 (inhibitory) and Ly-49D (activating) are highly homologous to Ly-49A and also recognize H-2Dd. However, the binding of Ly-49D and G2 to H-2Dd is of lower affinity than Ly-49A. All Ly-49s contain N-glycosylation motifs; however, the importance of receptor glycosylation in Ly-49-class I interactions has not been determined. Ly-49D and G2 contain a glycosylation motif (NTT (221–223)), absent in Ly-49A, adjacent to one of the proposed binding sites for H-2Dd (site 2). The presence of a complex carbohydrate group at this critical site could interfere with class I binding. In this study, we are able to demonstrate for the first time that Ly-49D binds H-2Dd in the presence of mouse β2-microglobulin. We also demonstrate that glycosylation of the NTT (221–23) motif of Ly-49D inteferes with recognition of H-2Dd. Alteration of the Ly-49D-NTT (221–23) motif to abolish glycosylation at this site resulted in enhanced H-2Dd binding and receptor activation. Furthermore, glycosylation of Ly-49G2 at NTT (221–23) also reduces receptor binding to H-2Dd tetramers. Therefore, the addition of complex carbohydrates to the Ly-49 family of receptors may represent a mechanism by which NK cells regulate affinity for host class I ligands.
Hemozoin Increases IFN-γ-Inducible Macrophage Nitric Oxide Generation Through Extracellular Signal-Regulated Kinase- and NF-κB-Dependent Pathways
Authors: Jaramillo Maritza , Gowda D. Channe , Radzioch Danuta , Olivier Martin
Published: April 2014

NO overproduction has been suggested to contribute to the immunopathology related to malaria infection. Even though a role for some parasite molecules (e.g., GPI) in NO induction has been proposed, the direct contribution of hemozoin (HZ), another parasite metabolite, remains to be established. Therefore, we were interested to determine whether Plasmodium falciparum (Pf) HZ and synthetic HZ, β-hematin, alone or in combination with IFN-γ, were able to induce macrophage (Mφ) NO synthesis. We observed that neither Pf HZ nor synthetic HZ led to NO generation in B10R murine Mφ; however, they significantly increased IFN-γ-mediated inducible NO synthase (iNOS) mRNA and protein expression, and NO production. Next, by investigating the transductional mechanisms involved in this cellular regulation, we established that HZ induces extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase phosphorylation as well as NF-κB binding to the iNOS promoter, and enhances the IFN-γ-dependent activation of both second messengers. Of interest, cell pretreatment with specific inhibitors against either NF-κB or the ERK1/2 pathway blocked the HZ + IFN-γ-inducible NF-κB activity and significantly reduced the HZ-dependent increase on IFN-γ-mediated iNOS and NO induction. Even though selective inhibition of the Janus kinase 2/STAT1α pathway suppressed NO synthesis in response to HZ + IFN-γ, HZ alone did not activate this signaling pathway and did not have an up-regulating effect on the IFN-γ-induced Janus kinase 2/STAT1α phosphorylation and STAT1α binding to the iNOS promoter. In conclusion, our results suggest that HZ exerts a potent synergistic effect on the IFN-γ-inducible NO generation in Mφ via ERK- and NF-κB-dependent pathways.
Repeated α-Galactosylceramide Administration Results in Expansion of NK T Cells and Alleviates Inflammatory Dermatitis in MRL-lpr/lpr Mice
Authors: Yang Jun-Qi , Saxena Vijay , Xu Honglin , Van Kaer Luc , Wang Chyung-Ru , Singh Ram Raj
Published: April 2014

NK T (NKT) cells expressing the invariant Vα14-Jα18 TCR α-chain recognize glycolipid Ags such as α-galactosylceramide (α-GalCer) presented by the MHC class I-like molecule CD1d. Upon activation by α-GalCer, invariant NKT cells secrete multiple cytokines and confer protection in certain immune-mediated disorders. Here we have investigated the role of NKT cells in the development of inflammatory dermatitis in MRL-lpr/lpr mice, which shares features with lupus in humans. Our results show that the numbers Sand functions of NKT (TCRβ+CD1d/α-GalCer tetramer+) cells, particularly of the NK1.1− subset, are reduced in MRL-lpr/lpr mice compared with MRL-fas/fas and/or nonautoimmune C3H/Hej and BALB/c mice. Repeated treatments with α-GalCer result in the expansion of NKT cells and alleviate dermatitis in MRL-lpr/lpr mice. Our results indicate that NKT cell deficiency can be corrected by repeated α-GalCer treatment and that NKT cells may play a protective role in inflammatory dermatitis of lupus-prone mice.
Cutting Edge: Dendritic Cell Actin Cytoskeletal Polarization during Immunological Synapse Formation Is Highly Antigen-Dependent
Authors: Al-Alwan Monther M. , Liwski Robert S. , Haeryfar S. M. Mansour , Baldridge William H. , Hoskin David W. , Rowden Geoffrey , West Kenneth A.
Published: April 2014

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4+ T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4+ T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.
Nitric Oxide Regulation of Human Peripheral Blood Mononuclear Cells: Critical Time Dependence and Selectivity for Cytokine versus Chemokine Expression
Authors: Macphail Sarah E. , Gibney Claire A. , Brooks Bernadette M. , Booth C. George , Flanagan Brian F. , Coleman John W.
Published: April 2014

NO is antiproliferative for T cells and other immune cells, but there is debate over whether it influences cytokine expression and if so whether it shows cytokine selectivity. Furthermore, the NO effect may depend on exposure time. To address these issues, we precultured human PBMC with the NO donors S-nitrosoglutathione (a natural storage form of NO) or S-nitroso-N-acetyl-d-penicillamine for up to 48 h before cell activation and then monitored proliferation and cytokine and chemokine expression. S-nitrosoglutathione or S-nitroso-N-acetyl-d-penicillamine, but not their non-NO-releasing analogues, inhibited proliferation induced by PHA or IL-2, the effect declining progressively from 48 to 0 h pre-exposure to the mitogen. This was accompanied by reduced PHA-induced IL-2 release and reduced IL-2, IFN-γ, and IL-13 mRNA expression. In contrast, NO did not influence PHA-induced expression of mRNA for the chemokines lymphotactin, RANTES, IFN-γ-inducible protein, macrophage-inhibitory protein-1α, macrophage-inhibitory protein-1β, macrophage chemoattractant protein-1, and IL-8 or release of RANTES or IL-8. The NO effects were not toxic and were not accompanied by changes in PHA-induced CD25 expression. We conclude that exposure time to NO is critical to altered PBMC responsiveness and that NO inhibits expression of both Th1 and Th2 cytokines but not chemokines.
The Phenotype of Inflammatory Macrophages Is Stimulus Dependent: Implications for the Nature of the Inflammatory Response
Authors: Cook Andrew D. , Braine Emma L. , Hamilton John A.
Published: April 2014

Many diseases are characterized by inflammatory reactions involving both the innate and adaptive arms of the immune system. Thioglycolate medium (TM) injection into the peritoneal cavity has long been used as a stimulus for eliciting inflammatory macrophages for study and for determining the importance of a particular mediator in inflammation. However, the response to this irritant may not be relevant to many inflammatory diseases. Therefore, we have developed an Ag-specific peritonitis model using methylated BSA (mBSA) as the stimulus. Priming mice intradermally with mBSA in adjuvant and boosting 14 days later, followed by an i.p. challenge with mBSA after an additional 7 days, led to an inflammatory reaction equivalent in magnitude to that induced with TM as judged by the number of exudate cells. The inflammatory macrophages elicited by the mBSA protocol differed, being smaller and less vacuolated than TM-elicited macrophages. Also, macrophages from 4-day mBSA-induced exudates expressed more MHC class II than TM-induced exudates, were able to stimulate allogeneic T lymphocytes, and upon in vitro stimulation with LPS secreted greater levels of IL-6 and IL-1β. Macrophages from 4-day TM-induced exudates, on the other hand, expressed Ly6C and ER-MP58, immature myeloid markers. The inflammatory response elicited using the Ag mBSA may be more relevant for studying the inflammatory responses in many diseases, such as those of autoimmune origin and those involving an acquired immune response.
Antibodies to Keyhole Limpet Hemocyanin Cross-React with an Epitope on the Polysaccharide Capsule of Cryptococcus neoformans and Other Carbohydrates: Implications for Vaccine Development
Authors: May Rena J. , Beenhouwer David O. , Scharff Matthew D.
Published: April 2014

Cryptococcus neoformans causes a life-threatening meningoencephalitis in AIDS patients. Mice immunized with a glycoconjugate vaccine composed of the glucuronoxylomannan (GXM) component of the cryptococcal capsular polysaccharide conjugated to tetanus toxoid produce Abs that can be either protective or nonprotective. Because nonprotective Abs block the efficacy of protective Abs, an effective vaccine must focus the Ab response on a protective epitope. Mice immunized with peptide mimetics of GXM conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde developed Abs to GXM. However, control peptides P315 and P24 conjugated to KLH also elicited Abs to GXM. GXM-binding Abs from mice immunized with P315-KLH were inhibited by KLH treated with glutaraldehyde (KLH-g), but not by P315. Furthermore, KLH-g inhibited binding of GXM by serum of mice immunized with GXM-TT, indicating that glutaraldehyde treatment of KLH reveals an epitope(s) that cross-reacts with GXM. Vaccination with KLH-g or unmodified KLH elicited Abs to GXM, but did not confer protection against C. neoformans, suggesting the cross-reactive epitope on KLH was not protective. This was supported by the finding that 4H3, a nonprotective mAb, cross-reacted strongly with KLH-g. Sera from mice immunized with either native KLH or KLH-g cross-reacted with several other carbohydrate Ags, many of which have been conjugated to KLH for vaccine development. This study illustrates how mAbs can be used to determine the efficacy of potential vaccines, in addition to describing the complexity of using KLH and glutaraldehyde in the development of vaccines to carbohydrate Ags.
Thymic CD8+ T Cell Production Strongly Influences Tumor Antigen Recognition and Age-Dependent Glioma Mortality
Authors: Wheeler Christopher J. , Black Keith L. , Liu Gentao , Ying Han , Yu John S. , Zhang Wenxuan , Lee Paul K.
Published: April 2014

For unknown reasons, advanced age remains a dominant predictor of poor clinical outcome for nearly all cancers. A decrease in the production of T cells by the thymus accompanies normal aging and parallels the age-dependent increase in cancer progression, but the specific impact of immunity on tumor progression in general is unknown. Glioblastoma multiforme (GBM), the most common primary brain neoplasm, is characterized by rapid age-dependent rates of progression and death. In this study, we show levels of CD8+ recent thymic emigrants (RTEs) accounted for the prognostic power of age on clinical outcome in GBM patients. CD8+ RTEs, typically a tiny proportion of CD8+ T cells, remarkably accounted for the majority of tumor Ag-binding small precursor cells in PBMC from these patients and from healthy individuals. Large blasting tumor Ag-binding cells comprised of CD8+ RTEs and phenotypically related cells were predominantly expanded following experimental vaccination of GBM patients. Quantification of CD8+ RTE expansion in vivo correlated strongly with vaccine-elicited cytokine responses, and estimated numbers of expanding CD8+ RTEs were consistent predictors of clinical outcome in vaccinated GBM patients. Targeted mutant (CD8β−/−) mice specifically deficient in thymic CD8+ T cell production uniquely displayed an age-specific decrease in glioma host survival as well as a strong correlation between host survival and thymus cellular production. These findings suggest that levels and function of newly produced CD8+ T cells critically influence age-dependent cancer mortality and exert one of the strongest known influences on GBM outcome by predominantly mediating clinically beneficial antitumor immune responses.
Tolerogenic APC Generate CD8+ T Regulatory Cells That Modulate Pulmonary Interstitial Fibrosis
Authors: Zhang-Hoover Jie , Stein-Streilein Joan
Published: April 2014

Transforming growth factor-β2-treated Ag-pulsed APC mimic APC from the immune privileged eye, and provide signals that generate regulatory T (Tr) cells and mediate peripheral tolerance. We postulated that TGF-β2-treated Ag-pulsed APC (tolerogenic APC (tol-APC)) might also orchestrate regulation of immune mediated pathogenesis in nonimmune privileged tissues such as the lung. We used an adoptive transfer model of autoimmune pulmonary interstitial fibrosis called hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) in this study. Mice that received 2,4,6-trinitrobenzene sulfonic acid-sensitized cells and challenged (intratracheally) with the hapten developed pulmonary interstitial fibrosis. However, transfer (i.v.) of TGF-β2-treated 2,4,6-trinitrobenzene sulfonic acid-pulsed bone marrow-derived APC (tol-APC) to experimental mice 1 day after intratracheal challenge reduced the collagen deposition in the interstitium of the lung that usually follows challenge. Furthermore, ADT-HIPIF mice that received tol-APC developed Ag-specific efferent CD8+ Tr cells. Adoptive transfer of the Tr cells to another set of presensitized mice mediated suppression of the efferent phase of Th1 immune response and the subsequent immune dependent pulmonary interstitial fibrosis. Thus, tol-APC induced efferent CD8+ Tr cells in immune mice, and the regulation of the immune response limited the development of autoimmune pulmonary fibrosis in sensitized and pulmonary-challenged mice. Because ADT-HIPIF shares etiological and pathological characteristics with a variety of human immune inflammatory conditions in the lung that eventuate into interstitial fibrosis, these studies provide insight into potential therapy to alter the course of pulmonary fibrosis in humans.
Essential Contribution of Monocyte Chemoattractant Protein-1/C-C Chemokine Ligand-2 to Resolution and Repair Processes in Acute Bacterial Pneumonia
Authors: Amano Hideaki , Morimoto Kounosuke , Senba Masachika , Wang Hui , Ishida Yuko , Kumatori Atsushi , Yoshimine Hiroyuki , Oishi Kazunori , Mukaida Naofumi , Nagatake Tsuyoshi
Published: April 2014

Neutrophil infiltration is the first step in eradication of bacterial infection, but neutrophils rapidly die after killing bacteria. Subsequent accumulation of macrophage lineage cells, such as alveolar macrophages (AMs), is essential to remove dying neutrophils, which are a source of injurious substances. Macrophage lineage cells can promote tissue repair, by producing potential growth factors including hepatocyte growth factor (HGF). However, it remains elusive which factor activates macrophage in these processes. Intratracheal instillation of Pseudomonas aeruginosa caused neutrophil infiltration in the airspace; subsequently, the numbers of total AMs and neutrophil ingested AMs were increased. Bronchoalveolar lavage (BAL) fluid levels of monocyte chemoattractant protein (MCP)-1/CC chemokine ligand-2 (CCL2), a potent macrophage-activating factor, were increased before the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid. Immunoreactive MCP-1 proteins were detected in alveolar type II epithelial cells and AMs only after P. aeruginosa infection. The administration of anti-MCP-1/CCL2 Abs reduced the increases in the number of AM-ingesting neutrophils and HGF levels in BAL fluid, and eventually aggravated lung tissue injury. In contrast, the administration of MCP-1/CCL2 enhanced the increases in the number of AM ingesting neutrophils and HGF levels in BAL fluid, and eventually attenuated lung tissue injury. Furthermore, MCP-1/CCL2 enhanced the ingestion of apoptotic neutrophils and HGF production by a mouse macrophage cell line, RAW 267.4, in a dose-dependent manner. Collectively, MCP-1/CCL2 has a crucial role in the resolution and repair processes of acute bacterial pneumonia by enhancing the removal of dying neutrophils and HGF production by AMs
Cytoskeletal Reorganization Internalizes Multiple Transient Receptor Potential Channels and Blocks Calcium Entry into Human Neutrophils
Authors: Itagaki Kiyoshi , Kannan Kolenkode B. , Singh Brij B. , Hauser Carl J.
Published: April 2014

Store-operated calcium entry (SOCE) is required for polymorphonuclear neutrophil (PMN) activation in response to G protein-coupled agonists. Some immunocytes express proteins homologous to the Drosophila transient receptor potential gene (trp) calcium channel. TRP proteins assemble into heterotetrameric ion channels and are known to support SOCE in overexpression systems, but the evidence that TRP proteins support SOCE and are functionally important in wild-type cells remains indirect. We therefore studied the expression and function of TRP proteins in primary human PMN. TRPC1, TRPC3, TRPC4, and TRPC6 were all expressed as mRNA as well as membrane proteins. Immunofluorescence microscopy demonstrated localization of TRPC1, TRPC3, and TRPC4 to the PMN cell membrane and their internalization after cytoskeletal reorganization by calyculin A (CalyA). Either TRPC internalization by CalyA or treatment with the inositol triphosphate receptor inhibitor 2-aminoethoxydiphenyl borane resulted in the loss of PMN SOCE. Cytochalasin D (CytoD) disrupts actin filaments, thus preventing cytoskeletal reorganization, and pretreatment with CytoD rescued PMN SOCE from inhibition by CalyA. Comparative studies of CytoD and 2-aminoethoxydiphenyl borane inhibition of PMN cationic entry after thapsigargin or platelet-activating factor suggested that SOCE occurs through both calcium-specific and nonspecific pathways. Taken together, these studies suggest that the multiple TRPC proteins expressed by human PMN participate in the formation of at least two store-operated calcium channels that have differing ionic permeabilities and regulatory characteristics.
Cutting Edge: C3d Functions as a Molecular Adjuvant in the Absence of CD21/35 Expression
Authors: Haas Karen M. , Toapanta Franklin R. , Oliver Julie A. , Poe Jonathan C. , Weis John H. , Karp David R. , Bower Joseph F. , Ross Ted M. , Tedder Thomas F.
Published: April 2014

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35−/−) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35−/− mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35−/− mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.
Depletion of CD8+ Cells Abolishes the Pregnancy Protective Effect of Progesterone Substitution with Dydrogesterone in Mice by Altering the Th1/Th2 Cytokine Profile
Authors: Blois Sandra M. , Joachim Ricarda , Kandil Judith , Margni Ricardo , Tometten Mareike , Klapp Burghard F. , Arck Petra C.
Published: April 2014

One of the most remarkable immunological regulations is the maternal immune tolerance toward the fetal semiallograft during pregnancy, which has been referred to as immunity’s pregnant pause. Rejection of the semiallogeneic trophoblast cells must be selectively inhibited and pathways presumably include Th2 cytokines unopposed by Th1 cytokines. Steroid hormones, including progesterone, have similar effects. Low levels of progesterone and Th2 cytokines and high levels of Th1 cytokines are attributable for increased abortions in mammalians, which may be triggered by psychoemotional stress. Thus, the aim of the present study was to provide experimental evidence for the mechanism involved in the mediation of immune responses by endocrine signals during pregnancy and stress-triggered pregnancy failure. DBA/2J-mated CBA/J female mice were randomized in three groups: 1) control females, 2) mice exposed to stress on gestation day 5.5, and 3) mice exposed to stress and substituted with dydrogesterone, a progestogen with a binding profile highly selective for the progesterone receptor on gestation day 5.5. On gestation days 7.5, 9.5, and 10.5, mice of each group were sacrificed, and the frequency of CD8+ cells and cytokine expression (IL-4, IL-12, TNF-α, IFN-γ) in blood and uterus cells was evaluated by flow cytometry. Additionally, some mice were depleted of CD8 cells by injection of mAb. We observed that progesterone substitution abrogated the abortogenic effects of stress exposure by decreasing the frequency of abortogenic cytokines. This pathway was exceedingly CD8-dependent, because depletion of CD8 led to a termination of the pregnancy protective effect of progesterone substitution.
Heart, but Not Skin, Allografts from Donors Lacking Flt3 Ligand Exhibit Markedly Prolonged Survival Time
Authors: Wang Zhiliang , Castellaneta Antonino , De Creus An , Shufesky William J. , Morelli Adrian E. , Thomson Angus W.
Published: April 2014

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L−/−) show severe reductions in both myeloid (CD11c+CD8α−) and lymphoid-related DC (CD11c+CD8α+) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c+ interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c+ cells that migrated from Flt3L−/− skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c+ cells. We examined the survival of Flt3L−/− heart or tail skin grafts (H2b) in allogeneic wt (BALB/c; H2d) recipients. The outcome of transplantation of BALB/c organs into Flt3L−/− recipients was also determined. Flt3L−/− mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L−/− heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L−/− hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L−/− hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L−/− recipients reflects presumably an intact role of the direct pathway of allorecognition.
IL-10-Secreting Regulatory T Cells Do Not Express Foxp3 but Have Comparable Regulatory Function to Naturally Occurring CD4+CD25+ Regulatory T Cells
Authors: Vieira Pedro L. , Christensen Jillian R. , Minaee Sophie , O’Neill Emma J. , Barrat Franck J. , Boonstra André , Barthlott Thomas , Stockinger Brigitta , Wraith David C. , O’Garra Anne
Published: April 2014

Regulatory T cells (TReg) control immune responses to self and nonself Ags. The relationship between Ag-driven IL-10-secreting TReg (IL-10-TReg) and naturally occurring CD4+CD25+ TReg is as yet unclear. We show that mouse IL-10-TReg obtained using either in vitro or in vivo regimens of antigenic stimulation did not express the CD4+CD25+ TReg-associated transcription factor Foxp3. However, despite the absence of Foxp3 expression, homogeneous populations of IL-10-TReg inhibited the in vitro proliferation of CD4+CD25− T cells with a similar efficiency to that of CD4+CD25+ TReg. This inhibition of T cell proliferation by IL-10-TReg was achieved through an IL-10-independent mechanism as seen for CD4+CD25+ TReg and was overcome by exogenous IL-2. Both IL-10-TReg and CD4+CD25+ TReg were similar in that they produced little to no IL-2. These data show that Foxp3 expression is not a prerequisite for IL-10-TReg activity in vitro or in vivo, and suggest that IL-10-TReg and naturally occurring CD4+CD25+ TReg may have distinct origins.