bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Neutralization of Human IL-6 (hIL-6) Achieved by Immunization of hIL-6-Transgenic Mice with a hIL-6 Receptor Antagonist
Authors: De Benedetti Fabrizio , Pignatti Patrizia , Vivarelli Marina , Meazza Cristina , Ciliberto Gennaro , Savino Rocco , Martini Alberto
Published: April 2014

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.
Endogenous Oocyte Antigens Are Required for Rapid Induction and Progression of Autoimmune Ovarian Disease Following Day-3 Thymectomy
Authors: Alard Pascale , Thompson Claire , Agersborg Sally S. , Thatte Jayant , Setiady Yulius , Samy Eileen , Tung Kenneth S. K.
Published: April 2014

Female (C57BL/6×A/J)F1 mice undergoing thymectomy on day 3 after birth (d3tx) developed autoimmune ovarian disease (AOD) and autoimmune disease of the lacrimal gland. As both were prevented by normal adult CD25+ T cells, regulatory T cell depletion is responsible for d3tx diseases. AOD began as oophoritis at 3 wk. By 4 wk, AOD progressed to ovarian atrophy with autoantibody response against multiple oocyte Ag of early ontogeny. The requirement for immunogenic endogenous ovarian Ag was investigated in d3tx female mice, d3tx male mice, and d3tx neonatally ovariectomized (OX) females. At 8 wk, all mice had comparable lacrimalitis but only those with endogenous ovaries developed AOD in ovarian grafts. The duration of Ag exposure required to initiate AOD was evaluated in d3tx mice OX at 2, 3, or 4 wk and engrafted with an ovary at 4, 5, or 6 wk, respectively. The mice OX at 2 wk did not have oophoritis whereas ∼80% of mice OX at 3 or 4 wk had maximal AOD, thus Ag stimulus for 2.5 wk following d3tx is sufficient. AOD progression requires additional endogenous Ag stimulation from the ovarian graft. In mice OX at 3 wk, ovaries engrafted at 5 wk had more severe oophoritis than ovaries engrafted at 6 or 12 wk; moreover, only mice engrafted at 5 wk developed ovarian atrophy and oocyte autoantibodies. Similar results were obtained in mice OX at 4 wk. Thus endogenous tissue Ag are critical in autoimmune disease induction and progression that occur spontaneously upon regulatory T cell depletion.
Diversity of the Killer Cell Ig-Like Receptors of Rhesus Monkeys
Authors: Hershberger Karen L. , Shyam Richa , Miura Ayako , Letvin Norman L.
Published: April 2014

Because the killer cell Ig-like receptors (KIRs) have only been characterized in humans and chimpanzees, we do not have a full understanding of their evolutionary history. Therefore, cDNAs encoding the KIR molecules of five rhesus monkeys were characterized, and were found to differ from the KIR molecules identified in humans and chimpanzees. Whereas only one KIR2DL4 molecule is detected in humans and chimpanzees, two distinct KIR2DL4 homologues were identified in the monkeys. Although the two human KIR3DL molecules are limited in their polymorphism, the KIR3DL homologues in the monkeys were highly polymorphic. Up to five KIR3DL homologues were identified in each monkey that was studied, and eleven distinct KIR3DL molecules were detected in the five rhesus monkeys. Two novel families of KIR molecules were identified in the rhesus monkeys, KIR3DH and KIR1D. The KIR3DH molecules have three Ig domains, transmembrane domains homologous to KIR2DL4 molecules that contain an arginine, and short cytoplasmic domains. With these features, the KIR3DH molecules resemble the activating forms of the human KIR molecules. The KIR1D molecule encodes only one complete Ig domain before a frame-shift in the second Ig domain occurs, leading to early termination of the molecule. Multiple splice variants of KIR1D exist that encode at least one Ig domain, as well as transmembrane and cytoplasmic domains. The extensive diversity of the rhesus monkey KIR3DL homologues and the novel KIR3DH and KIR1D molecules suggests that the KIR family of molecules has evolved rapidly during the evolution of primates.
NF-κB p50-Dependent In Vivo Footprints at Ig Sγ3 DNA Are Correlated with μ→γ3 Switch Recombination
Authors: Wuerffel Robert A. , Ma Limei , Kenter Amy L.
Published: April 2014

NF-κB has been demonstrated to play critical roles in multiple aspects of immune responses including Ig H chain isotype switching. To better define the specific roles the p50 subunit of NF-κB plays in μ→γ3 switch recombination (SR), we systematically evaluated p50-deficient B cells for activities that are strongly correlated with SR. B cell activation with LPS plus anti-IgD-dextran plus IL-5 plus IL-4 plus TGF-β produced normal levels of proliferation and γ3 germline transcripts in p50-deficient B cells, but μ→γ3 SR was impaired. In vitro binding studies previously showed that NF-κB p50 homodimer binds the switch nuclear B-site protein (SNIP) of the Sγ3 tandem repeat. Ligation-mediated PCR in vivo footprint analysis demonstrates that the region spanning the SNIP and switch nuclear A-site protein (SNAP) binding sites of the Sγ3 region are contacted by protein in normal resting splenic B cells. B cells that are homozygous for the targeted disruption of the gene encoding p50 (−/−) show strong aberrant footprints, whereas heterozygous cells (+/−) reveal a partial effect in Sγ3 DNA. These studies provide evidence of nucleoprotein interactions at switch DNA in vivo and suggest a direct interaction of p50 with Sγ3 DNA that is strongly correlated with SR competence.
Novel Androgen-Dependent Promoters Direct Expression of the C4b-Binding Protein α-Chain Gene in Epididymis
Authors: Nonaka Mayumi I. , Wang Guixian , Mori Takao , Okada Hidechika , Nonaka Masaru
Published: April 2014

C4b-binding protein (C4BP) is a large plasma protein composed of seven α-chains and one β-chain and is involved in the fluid phase regulation of the classical pathway of the complement system. Complement inhibitory activity is located in the α-chain, and its mRNA has been detected only in liver to date. Here, we have isolated cDNA clones encoding the α-chain of guinea pig C4BP (C4BPα) and have demonstrated significant C4BPα mRNA expression in epididymis as well as liver. The level of C4BPα transcripts increased in the epididymis after birth, while it remained constant in the liver. C4BPα mRNA was also detected in the normal murine epididymis at a significant level, but it decreased drastically after castration, suggesting that epididymal expression of the C4BPα gene is regulated by androgen. Gene analysis of guinea pig C4BPα indicated that liver and epididymis C4BPα mRNA share the coding region and 3′-untranslated region, but are transcribed from independent promoters on a single-copy gene. Two novel epididymis-specific promoters were identified in the region corresponding to the first intron of liver transcripts. The binding motif for hepatocyte NF-1 occurs in the promoter used for transcription of liver C4BPα, whereas androgen-responsive elements occur in both promoters used in the epididymis. These findings present a novel link between complement regulators and reproduction. Furthermore, variation in the 5′-untranslated regions, arising from alternative splicing of the newly identified exons, is demonstrable in the guinea pig C4BPα transcripts.
Granulocyte Macrophage-Colony-Stimulating Factor mRNA Is Stabilized in Airway Eosinophils and Peripheral Blood Eosinophils Activated by TNF-α Plus Fibronectin
Authors: Esnault Stéphane , Malter James S.
Published: April 2014

Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies have shown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controlling cytokine production remain obscure. We compared GM-CSF mRNA decay in eosinophils from bronchoalveolar lavage (BALEos) after allergen challenge or from PBEos. BALEos showed prolonged survival in vitro (60% at 4 days) and expressed GM-CSF mRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfection studies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin and TNF-α increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seen in BALEos. These data suggest that TNF-α plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSF production at a posttranscriptional level.
A Comparison of Mediators Released or Generated by IFN-γ-Treated Human Mast Cells Following Aggregation of FcγRI or FcεRI
Authors: Okayama Yoshimichi , Hagaman David D. , Metcalfe Dean D.
Published: April 2014

The high affinity receptor for IgG (FcγRI, CD64) is expressed on human mast cells, where it is up-regulated by IFN-γ and, thus, may allow mast cells to be recruited through IgG-dependent mechanisms in IFN-γ-rich tissue inflammation. However, the mediators produced by human mast cells after aggregation of FcγRI are incompletely described, and it is unknown whether these mediators are distinct from those produced after activation of human mast cells via FcεRI. Thus, we investigated the release of histamine and arachidonic acid metabolites and examined the chemokine and cytokine mRNA profiles of IFN-γ-treated cultured human mast cells after FcγRI or FcεRI aggregation. Aggregation of FcγRI resulted in histamine release and PGD2 and LTC4 generation. These responses were qualitatively indistinguishable from responses stimulated via FcεRI. Aggregation of FcεRI or FcγRI led to an induction or accumulation of 22 cytokine and chemokine mRNAs. Among them, seven cytokines (TNF-α, IL-1β, IL-5, IL-6, IL-13, IL-1R antagonist, and GM-CSF) were significantly up-regulated via aggregation of FcγRI compared with FcεRI. TNF-α mRNA data were confirmed by quantitative RT-PCR and ELISA. Furthermore, we confirmed histamine and TNF-α data using IFN-γ-treated purified human lung mast cells. Thus, aggregation of FcγRI on mast cells led to up-regulation and/or release of three important classes of mediators: biogenic amines, lipid mediators, and cytokines. Some cytokines, such as TNF-α, were released and generated to a greater degree after FcγRI aggregation, suggesting that selected biologic responses of mast cells may be preferentially generated through FcγRI in an IFN-γ-rich environment.
Cutting Edge: Telomerase Activation in Human T Lymphocytes Does Not Require Increase in Telomerase Reverse Transcriptase (hTERT) Protein But Is Associated with hTERT Phosphorylation and Nuclear Translocation
Authors: Liu Kebin , Hodes Richard J. , Weng Nan-ping
Published: April 2014

Capacity for cellular replication is critically important for lymphocyte function and can be regulated by telomerase-dependent maintenance of telomere length. In contrast to most normal human somatic cells that do not express telomerase due to the failure to transcribe telomerase reverse transcriptase (hTERT), lymphocytes express telomerase in a highly regulated fashion yet constitutively transcribe hTERT during development and activation. Here, we report that hTERT protein is present in both thymocytes and blood T cells at equivalent levels despite their substantial differences in telomerase activity, and that induction of telomerase activity in resting CD4+ T cells is not dependent on net hTERT protein increase. Moreover, hTERT is phosphorylated and translocated from cytoplasm to nucleus during CD4+ T cell activation. Thus, human T lymphocytes regulate telomerase function through novel events independent of hTERT protein levels, and hTERT phosphorylation and nuclear translocation may play a role in regulation of telomerase function in lymphocytes.
A CCR5-Dependent Novel Mechanism for Type 1 HIV gp120 Induced Loss of Macrophage Cell Surface CD4
Authors: Hewson Tim J. , Logie James J. , Simmonds Peter , Howie Sarah E. M.
Published: April 2014

Type 1 HIV gp120 is especially effective in disrupting immune cell function because it is able to cause dysregulation of both infected and uninfected cells. We report a novel CCR5-dependent mechanism of gp120-induced CD4 loss from macrophages. An M-tropic gp120, using CCR5, is able to induce 70% loss of cell surface CD4 from macrophages within an hour. This cell surface CD4 loss is more substantial and rapid than the 20% loss observed with T-tropic gp120IIIB by 3 h. The rapid and substantial CD4 loss induced by M-tropic gp120 is not observed on macrophages homozygous for the ccr5Δ32 mutation, which fail to express cell surface CCR5. We have used confocal imaging to show that gp120 and CD4 are internalized together by a process resembling receptor-mediated endocytosis, and that both proteins enter HLA-DR containing compartments of the macrophage. We have also shown by semiquantitative RT-PCR that, in response to CD4 loss from the cell surface, mRNA for CD4 is up-regulated and the intracellular pool of CD4 increases. CCR5 mRNA levels are also increased. It is proposed that internalization of self and viral protein and increased pools of intracellular CD4 could modulate Ag presentation efficiencies and have implications for the induction and maintenance of both productive immune responses and self-tolerance.
The Role of Dendritic Cells, B Cells, and M Cells in Gut-Oriented Immune Responses
Authors: Alpan Oral , Rudomen Gregory , Matzinger Polly
Published: April 2014

Although induction of T cell responses to fed Ag (oral tolerance) is thought to happen within the organized lymphoid tissue of the gut, we found that mice lacking Peyer’s patches, B cells, and the specialized Ag-handling M cells had no defect in the induction of T cell responses to fed Ag, whether assayed in vitro by T cell proliferation or cytokine production, or in vivo by delayed-type hypersensitivity or bystander suppression against mycobacterial Ags in CFA. Feeding of Ag had a major influence on dendritic cells from fed wild-type or μMT mice, such that these APCs were able to elicit a different class of response from naive T cells in vitro. These results suggest that systemic immune responses to soluble oral Ags do not require an organized gut-associated lymphoid tissue but are most likely induced by gut-conditioned dendritic cells that function both to initiate the gut-oriented response and to impart the characteristic features that discriminate it from responses induced parenterally.
Acute Rejection in the Absence of Cognate Recognition of Allograft by T Cells
Authors: Braun Michel Y. , Grandjean Isabelle , Feunou Pascal , Duban Livine , Kiss Robert , Goldman Michel , Lantz Olivier
Published: April 2014

We studied the effects of the indirect pathway of allograft recognition using T cells from TCR transgenic Marilyn mice, which recognize the male Ag H-Y in an I-Ab-restricted fashion. The T cells are not alloreactive to the H-2k haplotype, because they are not activated when adoptively transferred into recombinase-activating gene-2−/− common γ-chain−/− double-mutant H-2k male or female mice. However, skin from H-2k males, but not from H-2k females, is acutely rejected by recombinase-activating gene-2−/− transgenic female recipients. In vitro, Marylin spleen cells primed by H-2k skin grafting proliferated and secreted both IL-4 and IFN-γ in response to H-2k male stimulators. However, the removal of H-2b APC from the responding population abolished the response. Taken together, these results show that the indirect recognition that triggers rejection in this model is due to the recognition of H-Y Ag shed from H-2k male allograft and presented by the recipient’s own I-Ab APC to transgenic T cells. This study demonstrates unequivocally the capacity of naive CD4+ T cells to promote the rejection of allografts through mechanisms that involve indirect destruction of grafted tissues.
Antibody Binding to a Conformation-Dependent Epitope Induces L-Selectin Association with the Detergent-Resistant Cytoskeleton
Authors: Leid Jeff G. , Steeber Douglas A. , Tedder Thomas F. , Jutila Mark A.
Published: April 2014

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37°C, at 4°C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.
Adjuvanticity of α2-Macroglobulin, an Independent Ligand for the Heat Shock Protein Receptor CD91
Authors: Binder Robert J. , Karimeddini David , Srivastava Pramod K.
Published: April 2014

We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for α2-macroglobulin (α2M). Gp96 and α2M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8+ T cell responses and are re-presented by APCs, we tested α2M for similar properties. Our studies show that α2M binds peptides in vitro and that the peptides, chaperoned by α2M, efficiently prime peptide-specific CD8+ T cell responses in mice immunized with α2M-peptide complexes. Furthermore, peptides chaperoned by α2M, like those chaperoned by gp96, can be re-presented by CD91+ APCs on their MHC I molecules. These studies demonstrate that α2M molecules, like the heat shock protein molecules, are T cell adjuvants that can channel exogenous Ags into the endogenous pathway of Ag presentaion. The remarkable similarities between an intracellular chaperone and an extracellular serum chaperone may have interesting physiological ramifications.
The Activity of Immunoregulatory T Cells Mediating Active Tolerance Is Potentiated in Nonobese Diabetic Mice by an IL-4-Based Retroviral Gene Therapy
Authors: Yamamoto Ana Maria , Chernajovsky Yuti , Lepault Françoise , Podhajcer Osvaldo , Feldmann Marc , Bach Jean-François , Chatenoud Lucienne
Published: April 2014

Splenocytes from nonobese diabetic mice overexpressing murine IL (mIL)-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Diabetes appeared in 0–20% of mice injected with mIL-4-transduced cells vs 80–100% of controls injected with β-galactosidase-transduced cells. Protected mice showed a majority of islets (60%) presenting with noninvasive peri-insulitis at variance with β-galactosidase controls that exhibited invasive/destructive insulitis. Importantly, in all recipients, the transduced proteins were detected within islet infiltrates. Infiltrating lymphocytes from recipients of mIL-4-transduced cells produced high levels of mIL-4, as assessed by ELISA. In recipients of β-galactosidase-transduced cells, ∼60% of TCRαβ+ islet-infiltrating cells expressed β-galactosidase, as assessed by flow cytometry. The protection from disease transfer is due to a direct effect of mIL-4 gene therapy on immunoregulatory T cells rather than on diabetogenic cells. mIL-4-transduced purified CD62L− effector cells or transgenic BDC2.5 diabetogenic T cells still transferred disease efficiently. Conversely, mIL-4 transduction up-regulated the capacity of purified immunoregulatory CD62L+ cells to inhibit disease transfer. These data open new perspectives for gene therapy in insulin-dependent diabetes using T cells devoid of any intrinsic diabetogenic potential.
An HLA-DRB1-Derived Peptide Associated with Protection Against Rheumatoid Arthritis Is Naturally Processed by Human APCs
Authors: Snijders Alies , Elferink Diënne G. , Geluk Annemieke , van der Zanden A. Linda , Vos Koen , Schreuder Geziena M. T. , Breedveld Ferdinand C. , de Vries René R. P. , Zanelli Eric H.
Published: April 2014

Predisposition to rheumatoid arthritis (RA) is thought to be associated with HLA-DR1, -DR4, and -DR10. However, many epidemiological observations are better explained by a model in which the DQ alleles that are linked to these DR alleles, i.e., DQ5, DQ7, and DQ8, predispose to RA, while certain DR alleles have a dominant protective effect. All protective DRB1 alleles, e.g., *0402, *1301, and *1302, encode a unique motif, 70DERAA74. The protection may be explained by the presentation of DRB1-derived peptides by DQ to immunoregulatory T cells, because it was demonstrated in various autoimmune disease models that T cell responses to certain self-Ags can be involved in disease suppression. The aim of this study was to analyze whether peptides carrying the DERAA motif are naturally processed by human APC and presented in the context of the RA-predisposing DQ. Using a synthetic peptide carrying the DRB1*0402-derived sequence 65KDILEDERAAVDTYC79, we generated DERAA peptide-specific DQ-restricted T cell clones (TCC) from a DQ8 homozygous individual carrying DERAA-negative DR4 alleles. By analyzing the proliferation of these TCC, we demonstrated natural processing and presentation of the DERAA sequence by the APC of all the individuals (n = 12) carrying a DERAA-positive DRB1 allele and either DQ8 or the DQ8-related DQ7. Using a panel of truncated synthetic peptides, we identified the sequence 67(I)LEDERAAVD(TY)78 as the minimal determinant for binding to DQ8 and for recognition by the TCC. These findings support a model in which self-MHC-derived peptide can modulate predisposition to autoimmune disease in humans.
Murine Dendritic Cells Derived from Myeloid Progenitors of the Thymus Are Unable to Produce Bioactive IL-12p70
Authors: Martinon-Ego Catherine , Berthier Rolande , Cretin François , Collin Véronique , Laharie Anne-Marie , Marche Patrice N.
Published: April 2014

Dendritic cells (DC) are present at low density in the thymus where they mediate negative selection of self-reactive thymocytes. Previous reports suggest that thymic DC (TDC) are a single population of lymphoid-related DC. In this study, we documented the presence in the adult mouse thymus of an additional population of TDC exhibiting a myeloid phenotype (CD11c+ CD8α− CD11b+). This population, which can be purified, represented ∼20% of the total TDC and differs from the population of lymphoid TDC (CD11c+ CD8+ CD11b−) by its incapacity to produce IL-12p70 under double stimulation by LPS and anti-CD40. Furthermore, using an original culture system allowing expansion of DC from myeloid progenitors, we demonstrated that DC exhibiting a similar myeloid phenotype can be derived from a common DC/macrophage progenitor resident in the adult mouse thymus. We found that, in contrast with myeloid splenic DC expanded in the same conditions, these cultured TDC were unable to produce IL-12p70 under double stimulation by LPS and anti-CD40 or LPS and IFN-γ. Thus, our results suggest that 1) adult mouse thymus contains at least two phenotypically and functionally distinct populations of DC; and 2) cultured myeloid DC derived from thymus and spleen differ by their ability to produce IL-12p70. The mechanisms underlying the differences in IL-12-secreting capacities of the cultured splenic and thymic DC are under current investigation.
HLA-G2, -G3, and -G4 Isoforms Expressed as Nonmature Cell Surface Glycoproteins Inhibit NK and Antigen-Specific CTL Cytolysis
Authors: Riteau Béatrice , Rouas-Freiss Nathalie , Menier Catherine , Paul Pascale , Dausset Jean , Carosella Edgardo D.
Published: April 2014

HLA-G is a nonclassical MHC class I molecule that plays a major role in maternal-fetal tolerance. Four membrane-bound (HLA-G1 to -G4) and two soluble (HLA-G5, and -G6) proteins are generated by alternative splicing. Only HLA-G1 has been extensively studied in terms of both expression and function. We provide evidence here that HLA-G2, -G3, and -G4 truncated isoforms reach the cell surface of transfected cells, as endoglycosidase H-sensitive glycoproteins, after a 2-h chase period. Moreover, cytotoxicity experiments show that these transfected cells are protected from the lytic activity of both innate (NK cells) and acquired (CTL) effectors. These findings highlight the immunomodulatory role that HLA-G2, -G3, and -G4 proteins will assume during physiologic or pathologic processes in which HLA-G1 expression is altered.
Increased Transcription Levels Induce Higher Mutation Rates in a Hypermutating Cell Line
Authors: Bachl Jürgen , Carlson Chris , Gray-Schopfer Vanessa , Dessing Mark , Olsson Carina
Published: April 2014

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.
Role of the Actin Cytoskeleton in T Cell Absorption and Internalization of Ligands from APC
Authors: Hwang Inkyu , Sprent Jonathan
Published: April 2014

A feature of T-APC interaction is that, via either TCR or CD28, T cells can absorb molecules from APC on to the cell surface and then internalize these molecules. Here, using both normal and TCR-transgenic T cells, we investigated the mechanism of T cell absorption of molecules from APC and the role of the cytoskeleton. The results show that although activated T cells could absorb APC molecules in the form of cell fragments, uptake of molecules by resting T cells required direct T-APC interaction. Based on studies with latrunculin B, surface absorption of molecules by resting T cells was crucially dependent upon the actin cytoskeleton for both CD28- and TCR-mediated absorption. Significantly, however, TCR-mediated absorption became strongly resistant to latrunculin B when the concentration of MHC-bound peptide on APC was raised to a high level, implying that the actin cytoskeleton is only important for absorption when the density of receptor/ligand interaction is low. By contrast, in all situations tested, the actin cytoskeleton played a decisive role in controlling T cell internalization of ligands from the cell surface.
CD40 Signaling Converts a Minimally Immunogenic Antigen into a Potent Vaccine Against the Intracellular Pathogen Listeria monocytogenes
Authors: Rolph Michael S. , Kaufmann Stefan H. E.
Published: April 2014

Conventional vaccination strategies have failed for numerous pathogens, and the development of novel approaches to vaccine development is a major public health priority. Killed or subunit vaccines represent an attractive approach due to their safety, but they suffer from low immunogenicity and generally require adjuvants. In this study, the possibility of harnessing CD40 signaling for enhancing the immunogenicity of killed vaccines was investigated. Intravenous immunization of C57BL/6 mice with heat-killed Listeria monocytogenes (HKL) induced minimal immunity, but HKL administered together with an agonistic anti-CD40 mAb induced high levels of both CD4+ and CD8+ T cells capable of producing IFN-γ following in vitro HKL stimulation. HKL/anti-CD40 vaccination elicited robust protection against subsequent Listeria challenge. Approximately 1000-fold fewer bacteria were detected in the liver and spleen of vaccinated mice, and vaccinated mice were also able to resist a normally lethal Listeria challenge. CD40-mediated adjuvant activity required endogenous IL-12 at the time of vaccination, and protection was mediated by both CD8+ and CD4+ T cells. Thus, CD40 signaling can deliver potent adjuvant activity for vaccination against intracellular pathogens and is particularly effective for pathogens requiring both CD4+ and CD8+ T cells for effective control.
Regulatory Effects of Eotaxin on Acute Lung Inflammatory Injury
Authors: Guo Ren-Feng , Lentsch Alex B. , Warner Roscoe L. , Huber-Lang Markus , Sarma J. Vidya , Hlaing Tom , Shi Michael M. , Lukacs Nicholas W. , Ward Peter A.
Published: April 2014

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-κB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-α levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of 125I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1α, MIP-1β, TNF-α, and IL-1β. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1α, MIP-1β, TNF-α, and IL-1β expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.
Anergy Induction by Dimeric TCR Ligands
Authors: Appel Heiner , Seth Nilufer P. , Gauthier Laurent , Wucherpfennig Kai W.
Published: April 2014

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.
Cutting Edge: Antibody-Mediated Cessation of Hemotropic Infection by the Intraerythrocytic Mouse PathogenBartonella grahamii
Authors: Koesling Jan , Aebischer Toni , Falch Christine , Schülein Ralf , Dehio Christoph
Published: April 2014

The genus Bartonella includes important human-specific and zoonotic pathogens which cause intraerythrocytic bacteremia in their mammalian reservoir host(s). It is accepted that cellular immunity plays a decisive role in the host’s defense against most intracellular bacteria. Bartonella sp. infection in the immunocompetent host typically leads to immunity against homologous challenge. The basis of this immunity, be it cellular or humoral, is unclear. In this study, the course of Bartonella grahamii bacteremia in immunocompetent and immunocompromised mice was compared. In immunocompetent hosts, the bacteremia is transient and induces a strong humoral immune response. In contrast, bacteremia persists in immunocompromised B and T cell-deficient mice. Immune serum transfer beginning with day 6 postinfection to B cell-deficient mice unable to produce Igs converted the persistent bacteremia to a transient course indistinguishable from that of immunocompetent animals. These data demonstrate an essential role for specific Abs in abrogating the intraerythrocytic bacteremia of B. grahamii in mice.
CD28-independent Costimulation of T Cells in Alloimmune Responses
Authors: Yamada Akira , Kishimoto Koji , Dong Victor M. , Sho Masayuki , Salama Alan D. , Anosova Natalie G. , Benichou Gilles , Mandelbrot Didier A. , Sharpe Arlene H. , Turka Laurence A. , Auchincloss Hugh , Sayegh Mohamed H.
Published: April 2014

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4+ and CD8+ T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.
Cutting Edge: Germinal Centers Can Be Induced in the Absence of T Cells
Authors: Lentz Vicky M. , Manser Tim
Published: April 2014

Immunization of mice containing mutations that inactivate the TCR Cβ and Cδ genes with the T cell-independent (TI) type 2 Ag (4-hydroxy-3-nitrophenyl)acetyl-Ficoll induces clusters of peanut agglutinin-binding B cells in the spleen. These clusters are histologically indistinguishable from germinal centers (GCs) typical of T cell-dependent immune responses. They are located in follicles, and contain mature follicular dendritic cells, immune complex deposits, and B cells that display the phenotypic qualities of conventional GC B cells. However, the kinetics of this TI GC response differ from T cell-dependent GC responses in being rapidly induced and of short duration. Moreover, the Ab V genes expressed in TI GCs have not undergone somatic hypermutation. Therefore, T cells may be required for B cell differentiation processes associated with the intermediate and latter stages of the GC reaction, but they are dispensable for the induction and initial development of this response.
TNF-α-Induced Sphingosine 1-Phosphate Inhibits Apoptosis Through a Phosphatidylinositol 3-Kinase/Akt Pathway in Human Hepatocytes
Authors: Osawa Yosuke , Banno Yoshiko , Nagaki Masahito , Brenner David A. , Naiki Takafumi , Nozawa Yoshinori , Nakashima Shigeru , Moriwaki Hisataka
Published: April 2014

Human hepatocytes usually are resistant to TNF-α cytotoxicity. In mouse or rat hepatocytes, repression of NF-κB activation is sufficient to induce TNF-α-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IκBα (Ad5IκB), which almost completely blocks NF-κB activation, >80% of the cells survived 24 h after TNF-α stimulation. Here, we report that TNF-α activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-α in Ad5IκB-infected Huh-7 and Hc cells. TNF-α-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-α-induced apoptosis. Although Akt has been reported to activate NF-κB, DMS and LY 294002 failed to prevent TNF-α-induced NF-κB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-κB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-κB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-α and probably FasL.
Generation of Anergic and Potentially Immunoregulatory CD25+CD4 T Cells In Vivo After Induction of Peripheral Tolerance with Intravenous or Oral Antigen
Authors: Thorstenson Kristen M. , Khoruts Alexander
Published: April 2014

Immunoregulatory CD25+CD4 T cells are thought to arise from the thymus as a distinct lineage of CD4 T cells specific for self Ags. We used the DO11.10 TCR transgenic adoptive transfer system to show that cells of similar phenotype may also arise in the course of peripheral tolerance induction. Such cells emerged within 1 wk following Ag exposure and correlated negatively with the number of initial cell divisions. Limiting i.v. Ag dose or using an oral tolerance protocol yielded the greatest numbers of Ag-specific CD25+CD4 T cells. In contrast, immunogenic Ag exposure in the presence of an adjuvant did not lead to emergence of CD25+CD4 T cells. The profound anergic phenotype of these cells and their potential immunoregulatory properties make them an especially desirable population to induce in the course of immunotherapy in numerous clinical settings. This experimental system may be useful in future studies designed to optimize immunologic tolerance induction.
Rapid Induction of Naive T Cell Apoptosis by Ecto-Nicotinamide Adenine Dinucleotide: Requirement for Mono(ADP-Ribosyl)Transferase 2 and a Downstream Effector
Authors: Adriouch Sahil , Ohlrogge Wiebke , Haag Friedrich , Koch-Nolte Friedrich , Seman Michel
Published: April 2014

Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC50 of 3–5 μM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [32P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [32P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 × NZW)F1 animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 × NZW)F1 T cells. The fact that T cells of F1 animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).
Transcription Factor NF-κB Regulates Igλ Light Chain Gene Rearrangement
Authors: Bendall Heather H. , Sikes Michael L. , Oltz Eugene M.
Published: April 2014

The tissue- and stage-specific assembly of Ig and TCR genes is mediated by a common V(D)J recombinase complex in precursor lymphocytes. Directed alterations in the accessibility of V, D, and J gene segments target the recombinase to specific Ag receptor loci. Accessibility within a given locus is regulated by the functional interaction of transcription factors with cognate enhancer elements and correlates with the transcriptional activity of unrearranged gene segments. As demonstrated in our prior studies, rearrangement of the Igκ locus is regulated by the inducible transcription factor NF-κB. In contrast to the Igκ locus, known transcriptional control elements in the Igλ locus lack functional NF-κB binding sites. Consistent with this observation, the expression of assembled Igλ genes in mature B cells has been shown to be NF-κB independent. Nonetheless, we now show that specific repression of NF-κB inhibits germline transcription and recombination of Igλ gene segments in precursor B cells. Molecular analyses indicate that the block in NF-κB impairs Igλ rearrangement at the level of recombinase accessibility. In contrast, the activities of known Igλ promoter and enhancer elements are unaffected in the same cellular background. These findings expand the range of NF-κB action in precursor B cells beyond Igκ to include the control of recombinational accessibility at both L chain loci. Moreover, our results strongly suggest the existence of a novel Igλ regulatory element that is either directly or indirectly activated by NF-κB during the early stages of B cell development.
Critical Role for CD8 in Binding of MHC Tetramers to TCR: CD8 Antibodies Block Specific Binding of Human Tumor- Specific MHC-Peptide Tetramers to TCR
Authors: Denkberg Galit , Cohen Cyril J. , Reiter Yoram
Published: April 2014

There are conflicting opinions about the role that the T cell coreceptors CD4 and CD8 play in TCR binding and activation. Recent evidence from transgenic mouse models suggests that CD8 plays a critical role in TCR binding and activation by peptide-MHC complex multimers (tetramers). Here we show with a human CTL clone specific for a tumor-associated MHC-peptide complex that the binding of tetramers to the TCR on these cells is completely blocked by anti-human CD8 Abs. Moreover, the staining of CTLs with specific MHC-peptide tetramers simultaneously with anti-CD8 Abs was completely blocked with three different anti-CD8 Abs. This blockage was mediated by anti-CD8 Abs but not anti-CD3 Abs and was dose dependent. The blocking effect of the anti-CD8 Abs was attributable to directly inhibiting tetramer binding and was not attributable to Ab-mediated TCR-CD8 internalization and down-regulation. Our results have important implications in TCR binding to MHC-peptide tetramers. MHC-peptide tetramers are widely used today in combination with anti-CD8 Abs for the phenotypic analysis of T cell populations and in the study of T cell responses under various pathological conditions such as infectious diseases and cancer. Our results indicate that also in the human system CD8 plays a critical role in the interaction of MHC-peptide multimers with TCR.
The Sushi Domain of Soluble IL-15 Receptor α Is Essential for Binding IL-15 and Inhibiting Inflammatory and Allogenic Responses In Vitro and In Vivo
Authors: Wei Xiao-qing , Orchardson Michael , Gracie J. Alastair , Leung Bernard P. , Gao Bao-mei , Guan Hui , Niedbala Wanda , Paterson Gavin K. , McInnes Iain B. , Liew Foo Y.
Published: April 2014

IL-15 is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 functions through the trimeric IL-15R complex, which consists of a high affinity binding α-chain and the common IL-2R β- and γ-chains. Characterization of IL-15/IL-15R interactions may facilitate the development of improved IL-15 antagonists for therapeutic interventions. We previously constructed soluble murine IL-15Rα (sIL-15Rα) by deleting the cytoplasmic and transmembrane domains. To localize the functional domain of IL-15Rα, we have now constructed various truncated versions of sIL-15Rα. The shortest region retaining IL-15 binding activity is a 65-aa sequence spanning the Sushi domain of IL-15Rα. Sushi domains, common motifs in protein-protein interactions, contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern. Amino acid substitution of the first or fourth cysteine in sIL-15Rα completely abolished its IL-15 binding activity. This also abrogated the ability of sIL-15Rα to neutralize IL-15-induced proinflammatory cytokine production and anti-apoptotic response in vitro. Furthermore, the mutant sIL-15Rα lost its ability to inhibit carrageenan-induced local inflammation and allogenic cell-induced T cell proliferation and cytokine production in vivo. Thus, the Sushi domain is critical for the functional activity of sIL-15Rα.