bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependency of Direct Pathway CD4+ T Cells on CD40-CD154 Costimulation Is Determined by Nature and Microenvironment of Primary Contact with Alloantigen
Authors: van Maurik Andre , de St. Groth Barbara Fazekas , Wood Kathryn J. , Jones Nick D.
Published: April 2014

Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4+ T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4+ T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4+ T cells that are reactive to the MHC class II alloantigen, H2As. An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2As-reactive CD4+ T cells when challenged with different forms of H2As+ alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an i.v. infusion of H2As+ leukocytes in combination with anti-CD154 therapy rapidly deletes H2As-reactive CD4+ T cells. In contrast, following transplantation of an H2As+ cardiac allograft, H2As-reactive CD4+ T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4+ T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.
A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions
Authors: Whitten Miranda M. A. , Tew Ian F. , Lee Bok L. , Ratcliffe Norman A.
Published: April 2014

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and β-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of β-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.
Extremely Rapid and Intense Induction of Apoptosis in Human Eosinophils by Anti-CD30 Antibody Treatment In Vitro
Authors: Matsumoto Kenji , Terakawa Maki , Miura Kenju , Fukuda Shuhei , Nakajima Toshiharu , Saito Hirohisa
Published: April 2014

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin’s lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab′)2 of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-κB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.
The Cytoplasmic Domain of Igα Is Necessary and Sufficient to Support Efficient Early B Cell Development
Authors: Pike Kelly A. , Iacampo Sandra , Friedmann Jennifer E. , Ratcliffe Michael J. H.
Published: April 2014

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igα and Igβ are expressed by fusion with the extracellular and transmembrane domains of murine CD8α and CD8β, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igα, in the absence of the cytoplasmic domain of Igβ, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igβ in the absence of the cytoplasmic domain of Igα failed to support B cell development. The ability of the cytoplasmic domain of Igα to support early B cell development required a functional Igα immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igα to the B cell surface, thereby initiating subsequent stages of development.
HIV-Infected Langerhans Cells Preferentially Transmit Virus to Proliferating Autologous CD4+ Memory T Cells Located within Langerhans Cell-T Cell Clusters
Authors: Sugaya Makoto , Loré Karin , Koup Richard A. , Douek Daniel C. , Blauvelt Andrew
Published: April 2014

Langerhans cells (LC) are likely initial targets for HIV following sexual exposure to virus and provide an efficient means for HIV to gain access to lymph node T cells. The purpose of this study was to examine the nature of the CD4+ T cell that becomes infected by HIV-infected LC. We infected human LC within tissue explants ex vivo and then, 3 days later, cocultured HIV-infected LC with different subsets of autologous CD4+ T cells. Using multicolor flow cytometric analyses of LC-CD4+ T cell cocultures, we documented that HIV-infected LC preferentially infected memory (as compared with naive) CD4+ T cells. Proliferating and HIV-infected CD4+ memory T cells were more frequently detected in conjugates of LC and autologous CD4+ T cells, suggesting that T cells become activated and preferentially get infected through cluster formation with infected LC, rather than getting infected with free virus produced by single HIV-infected LC or T cells. p24+ Memory CD4+ T cells proliferated well in the absence of superantigen; by contrast, p24+ T cells did not divide or divided only once in the presence of staphylococcal enterotoxin B, suggesting that virus production was rapid and induced apoptosis in these cells before significant proliferation could occur. These results highlight that close interactions between dendritic cells, in this case epidermal LC, and T cells are important for optimal HIV replication within specific subsets of CD4+ T cells. Disrupting cluster formation between LC and memory CD4+ T cells may be a novel strategy to interfere with sexual transmission of HIV.
WSX-1 and Glycoprotein 130 Constitute a Signal-Transducing Receptor for IL-27
Authors: Pflanz Stefan , Hibbert Linda , Mattson Jeanine , Rosales Rency , Vaisberg Elena , Bazan J. Fernando , Phillips Joseph H. , McClanahan Terrill K. , de Waal Malefyt Rene , Kastelein Robert A.
Published: April 2014

The recently discovered cytokine IL-27 belongs to the IL-6/IL-12 family of cytokines and induced proliferation of naive CD4+ T cells and the generation of a Th1-type adaptive immune response. Although binding of IL-27 to the cytokine receptor WSX-1 was demonstrated, this interaction proved insufficient to mediate cellular effects. Hence, IL-27 was believed to form a heteromeric signaling receptor complex with WSX-1 and another, yet to be identified, cytokine receptor subunit. In this study, we describe that WSX-1 together with gp130 constitutes a functional signal-transducing receptor for IL-27. We show that neither of the two subunits itself is sufficient to mediate IL-27-induced signal transduction, but that the combination of both is required for this event. Expression analysis of WSX-1 and gp130 by quantitative PCR suggests that IL-27 might have a variety of cellular targets besides naive CD4+ T cells: we demonstrate gene induction of a subset of inflammatory cytokines in primary human mast cells and monocytes in response to IL-27 stimulation. Thus, IL-27 not only contributes to the development of an adaptive immune response through its action on CD4+ T cells, it also directly acts on cells of the innate immune system.
Antigen Nonspecific Suppression of T Cell Responses by Activated Stimulation-Refractory CD4+ T Cells
Authors: Duthoit Christine T. , Nguyen Phuong , Geiger Terrence L.
Published: April 2014

Several classes of anergic T cells are capable of suppressing naive T cell proliferation and thereby limiting immune responses. Activated T cells, although not anergic, are transiently refractory to restimulation with Ag. We examine in this study whether activated refractory murine T cells can also suppress naive T cell responses. We find that they can, and that they exhibit many of the suppressive properties of anergic T cells. The activated cells strongly diminish Ag-mediated T cell proliferation, an activity that correlates with their refractory period. Suppression is independent of APC numbers and requires cell contact or proximity. Naive T cells stimulated in the presence of activated refractory cells up-regulate CD25 and CD69, but fail to produce IL-2. The addition of IL-2 to culture medium, however, does not prevent the suppression, which is therefore not solely due to the absence of this growth factor. Persistence of the suppressor cells is also not essential. T cells stimulated in their presence and then isolated from them and cultured do not divide. The suppressive cells, however, do not confer a refractory or anergic state on the target T lymphocytes, which can fully respond to antigenic stimulation if removed from the suppressors. Our results therefore provide evidence that activated T cells act as transient suppressor cells, severely constraining bystander T cell stimulation and thereby restricting their response. These results have potentially broad implications for the development and regulation of immune responses.
The Impact of Thymic Antigen Diversity on the Size of the Selected T Cell Repertoire
Authors: Faro Jose , Velasco Santiago , González-Fernández África , Bandeira Antonio
Published: April 2014

The TCR repertoire of a normal animal is shaped in the thymus by ligand-specific positive- and negative-selection events. These processes are believed to be determined at the single-cell level primarily by the affinity of the TCR-ligand interactions. The relationships among all the variables involved are still unknown due to the complexity of the interactions and the lack of quantitative analysis of those parameters. In this study, we developed a quantitative model of thymic selection that provides estimates of the fractions of positively and negatively selected thymocytes in the cortex and in the medulla, as well as upper-bound ranges for the number of selecting ligands required for the generation of a normal diverse TCR repertoire. Fitting the model to current estimates of positive- and negative-selected thymocytes leads to specific predictions. The results indicate the following: 1) the bulk of thymocyte death takes place in the cortex, and it is due to neglect; 2) the probability of a thymocyte to be negatively selected in the cortex is at least 10-fold lower than in the medulla; 3) <60 ligands are involved in cortical positive selection; and 4) negative selection in the medulla is constrained by a large diversity of selecting ligands on medullary APCs.
Human Cytomegalovirus Proteins pp65 and Immediate Early Protein 1 Are Common Targets for CD8+ T Cell Responses in Children with Congenital or Postnatal Human Cytomegalovirus Infection
Authors: Gibson Laura , Piccinini Giampiero , Lilleri Daniele , Revello Maria Grazia , Wang Zhongde , Markel Susan , Diamond Don J. , Luzuriaga Katherine
Published: April 2014

Recombinant modified vaccinia Ankara- and peptide-based IFN-γ ELISPOT assays were used to detect and measure human CMV (HCMV)-specific CD8+ T cell responses to the pp65 (UL83) and immediate early protein 1 (IE1; UL123) gene products in 16 HCMV-infected infants and children. Age at study ranged from birth to 2 years. HCMV-specific CD8+ T cells were detected in 14 (88%) of 16 children at frequencies ranging from 60 to >2000 spots/million PBMC. Responses were detected as early as 1 day of age in infants with documented congenital infection. Nine children responded to both pp65 and IE1, whereas responses to pp65 or IE1 alone were detected in three and two children, respectively. Regardless of the specificity of initial responses, IE1-specific responses predominated by 1 year of age. Changes in HCMV epitopes targeted by the CD8+ T cell responses were observed over time; epitopes commonly recognized by HLA-A2+ adults with latent HCMV infection did not fully account for responses detected in early childhood. Finally, the detection of HCMV-specific CD8+ T cell responses was temporally associated with a decrease in peripheral blood HCMV load. Taken altogether, these data demonstrate that the fetus and young infant can generate virus-specific CD8+ T cell responses. Changes observed in the protein and epitope-specificity of HCMV-specific CD8+ T cells over time are consistent with those observed after other primary viral infections. The temporal association between the detection of HCMV-specific CD8+ T cell responses and the reduction in blood HCMV load supports the importance of CD8+ T cells in controlling primary HCMV viremia.
NF-κB Controls Cell Fate Specification, Survival, and Molecular Differentiation of Immunoregulatory Natural T Lymphocytes
Authors: Stanic Aleksandar K. , Bezbradica Jelena S. , Park Jang-June , Matsuki Naoto , Mora Ana L. , Van Kaer Luc , Boothby Mark R. , Joyce Sebastian
Published: April 2014

Ontogenetic, homeostatic, and functional deficiencies within immunoregulatory natural T (iNKT) lymphocytes underlie various inflammatory immune disorders including autoimmunity. Signaling events that control cell fate specification and molecular differentiation of iNKT cells are only partly understood. Here we demonstrate that these processes within iNKT cells require classical NF-κB signaling. Inhibition of NF-κB signaling blocks iNKT cell ontogeny at an immature stage and reveals an apparent, novel precursor in which negative selection occurs. Most importantly, this block occurs due to a lack of survival signals, as Bcl-xL overexpression rescues iNKT cell ontogeny. Maturation of immature iNKT cell precursors induces Bcl-2 expression, which is defective in the absence of NF-κB signaling. Bcl-xL overexpression also rescues this maturation-induced Bcl-2 expression. Thus, antiapoptotic signals relayed by NF-κB critically control cell fate specification and molecular differentiation of iNKT cells and, hence, reveal a novel role for such signals within the immune system.
Adjuvant Costimulation during Secondary Antigen Challenge Directs Qualitative Aspects of Oral Tolerance Induction, Particularly during the Neonatal Period
Authors: Tobagus Iriani T. , Thomas Wayne R. , Holt Patrick G.
Published: April 2014

In this report we demonstrate that although passive feeding of specific Ag to mice as neonates or adults can induce oral tolerance in both the cellular and humoral arms of the immune response, quantitative and, in particular, qualitative aspects of the tolerance process are determined by the nature of the inflammatory costimuli provided at the time of secondary Ag challenge. Moreover, this dependency upon nonspecific costimulation is more profound in Ag-fed neonates than in their adult counterparts. Thus, administration of Ag in the Th1-selective adjuvant CFA to prefed animals resulted in significant inhibition of IgG2a, IL-2, and IFN-γ responses, whereas IL-5 responses were increased. In contrast, rechallenge with Ag in the Th2-selective adjuvant aluminum hydroxide resulted in significant inhibition of IgG1, IgE, IL-2, and IL-5 responses, whereas IFN-γ responses were increased. Additionally, although soluble Ag challenge of prefed adults revealed marginal tolerogenic effects, the same challenge protocol in animals prefed as neonates elicited enhanced Th2-dependent IgG1 production. These results suggest that inflammatory stimulation at the time of Ag challenge is obligatory to trigger oral tolerance mechanisms, particularly in animals fed as neonates and also that the type of adjuvant used at the time of challenge selects for the type of Th cell population to be inhibited.
CD4+ T Cells Regulate CD8+ T Cell-Mediated Cutaneous Immune Responses by Restricting Effector T Cell Development through a Fas Ligand-Dependent Mechanism
Authors: Gorbachev Anton V. , Fairchild Robert L.
Published: April 2014

The magnitude and duration of CD8+ T cell-mediated responses in the skin to hapten sensitization and challenge, contact hypersensitivity (CHS), is negatively regulated by CD4+ T cells through an unknown mechanism. In this study we show that CD4+ T cells restrict the development and expansion of hapten-specific CD8+ T cells mediating CHS responses to 2,4-dinitrofluorobenzene. In the absence of CD4+ T cells, high numbers of hapten-specific CD8+ T cells producing IFN-γ were detected in the skin-draining lymph nodes on day 5 postsensitization, and these numbers decreased slightly, but were maintained through day 9, correlating with the increased magnitude and duration of CHS responses observed in these mice. In the presence of CD4+ T cells, the number of hapten-specific CD8+ T cells producing IFN-γ detected on day 5 postsensitization was lower and quickly fell to background levels by day 7. The limited development of effector CD8+ T cells was associated with decreased numbers of hapten-presenting dendritic cells in the lymphoid priming site. This form of immunoregulation was absent after sensitization of Fas ligand-defective gld mice. Transfer of wild-type CD4+ T cells to gld mice restored the negative regulation of CD8+ T cell priming and the immune response to hapten challenge in gld-recipient mice. These results indicate that CD4+ T cells restrict hapten-specific CD8+ T cell priming for CHS responses through a Fas ligand-dependent mechanism.
Reduced Thymocyte Development in Sonic Hedgehog Knockout Embryos
Authors: Shah Divya K. , Hager-Theodorides Ariadne L. , Outram Susan V. , Ross Susan E. , Varas Alberto , Crompton Tessa
Published: April 2014

The Hedgehog family of secreted intercellular signaling molecules are regulators of patterning and organogenesis during animal development. In this study we provide genetic evidence that Sonic Hedgehog (Shh) has a role in the control of murine T cell development. Analysis of Shh−/− mouse embryos revealed that Shh regulates fetal thymus cellularity and thymocyte differentiation. Shh is necessary for expansion of CD4−CD8− double-negative (DN) thymocytes and for efficient transition from the earliest CD44+CD25− DN population to the subsequent CD44+CD25+ DN population and from DN to CD4+CD8+ double-positive cells.
The Impact of Self-Tolerance on the Polyclonal CD8+ T Cell Repertoire
Authors: Kessels Helmut W. H. G. , de Visser Karin E. , Tirion Felicia H. , Coccoris Miriam , Kruisbeek Ada M. , Schumacher Ton N. M.
Published: April 2014

TCRs possess considerable cross-reactivity toward structurally related Ags. Because the signaling threshold for negative selection is lower than that required for activation of mature T cells, the question arises as to which extent thymic deletion of self-specific T cells affects T cell responsiveness toward foreign peptides. In this study we show, in three different mouse models systems, that the polyclonal CD8+ T cell repertoire has a marked ability to react against the majority of Ags related to self despite self-tolerance, even in cases where self and foreign differ only marginally at a single TCR-contact residue. Thus, while individual T cells are markedly cross-reactive, the ability to distinguish between closely related Ags is introduced at the polyclonal T cell level.
Immunosuppressive Activity of Endovanilloids:N-Arachidonoyl-Dopamine Inhibits Activation of the NF-κB, NFAT, and Activator Protein 1 Signaling Pathways
Authors: Sancho Rocío , Macho Antonio , de La Vega Laureano , Calzado Marco A. , Fiebich Bernd L. , Appendino Giovanni , Muñoz Eduardo
Published: April 2014

Endogenous N-acyl dopamines such as N-arachidonoyldopamine (NADA) and N-oleoyldopamine have been recently identified as a new class of brain neurotransmitters sharing endocannabinoid and endovanilloid biological activities. As endocannabinoids show immunomodulatory activity, and T cells play a key role in the onset of several diseases that affect the CNS, we have evaluated the immunosuppressive activity of NADA and N-oleoyldopamine in human T cells, discovering that both compounds are potent inhibitors of early and late events in TCR-mediated T cell activation. Moreover, we found that NADA specifically inhibited both IL-2 and TNF-α gene transcription in stimulated Jurkat T cells. To further characterize the inhibitory mechanisms of NADA at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-κB, NF-AT, and AP-1 transcription factors in Jurkat cells. We found that NADA inhibited NF-κB-dependent transcriptional activity without affecting either degradation of the cytoplasmic NF-κB inhibitory protein, IκBα, or DNA binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by NADA in stimulated cells. In addition, NADA inhibited both binding to DNA and the transcriptional activity of NF-AT and AP-1, as expected from the inhibition of NF-AT1 dephosphorylation and c-Jun N-terminal kinase activation in stimulated T cells. Finally, overexpression of a constitutively active form of calcineurin demonstrated that this phosphatase may represent one of the main targets of NADA. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases.
Biases in Ig λ Light Chain Rearrangements in Human Intestinal Plasma Cells
Authors: Su Wen , Boursier Laurent , Padala Archana , Sanderson Jeremy D. , Spencer Jo
Published: April 2014

Human intestinal lamina propria plasma cells are considered to be the progeny of chronically stimulated germinal centers located in organized gut-associated lymphoid tissues such as Peyer’s patches and isolated lymphoid follicles. We have sampled human colonic lamina propria plasma cells and naive and memory B cell subsets from human Peyer’s patches by microdissection of immunohistochemically stained tissue sections and used PCR methods and sequence analysis to compare IgVλJλ rearrangements in the plasma cell and B cell populations. Rearrangements that were either in-frame or out-of-frame between V and J were compared. Usage of IgVλ families in the in-frame rearrangements from the plasma cells resembled that observed in the mantle cells, suggesting that antigenic selection for cellular specificity does not dramatically favor any particular Vλ segment. However, in marked contrast, out-of-frame rearrangements involving Vλ1 and Vλ2 families are rarely observed in intestinal plasma cells, whereas rearrangements involving Vλ5 are increased. This resulted in significantly biased ratios of in-frame:out-of-frame rearrangements in these Vλ families. Out-of-frame rearrangements of IgVλJλ from plasma cells, including those involving the Vλ5 family, have a significant tendency not to involve Jλ1, consistent with the hypothesis that this population includes rearrangements generated by secondary recombination events. We propose that modification of out-of-frame rearrangements of IgVλJλ exists, probably a consequence of secondary rearrangements. This may be a mechanism to avoid translocations to susceptible out-of-frame IgVλJλ rearrangements during somatic hypermutation.
Conformation and Glycosylation of a Megalin Fragment Correlate with Nephritogenicity in Heymann Nephritis
Authors: Tramontano Alfonso , Makker Sudesh P.
Published: April 2014

Active Heymann nephritis (AHN), a rat model of autoimmune glomerulonephritis, is induced by immunization with autologous megalin, a 600-kDa cell surface glycoprotein isolated from crude renal extracts. Recombinant proteins containing a 563-residue N-terminal sequence of megalin were obtained from Escherichia coli and baculovirus-insect cell expression systems. Rats immunized with the soluble, secreted protein encoded by a baculovirus construct elicited high titer anti-megalin autoantibodies and developed glomerular immune deposits and elevated proteinuria consistent with AHN. Rats treated with the bacterial or nonsecreted insect cell proteins produced a milder anti-megalin response and did not develop the disease. Nephritogenicity appeared to correlate with conformational or other structural features of native megalin. All three recombinant proteins were reactive in Western blots with rabbit anti-megalin antiserum, whereas the insect cell-derived proteins reacted preferentially in Western blot and ELISA with anti-megalin autoantibodies from rats with AHN induced by native megalin. Only the secreted insect cell product was stained in a lectin blot, suggesting its specific glycosylation. These observations provide evidence that a megalin N-terminal domain includes B and T cell epitopes sufficient for a pathogenic autoimmune response and that a native-like conformation and glycosylation are essential for the induction of disease. The importance of conformational B cell epitopes for pathogenic autoantibodies recapitulates observations made in other models of organ-specific autoimmune disease. Glycosidic modifications could influence the presentation of either B or T cell epitopes in AHN, consistent with emerging evidence of the role of post-translational modifications in pathogenic autoimmune responses.
The Crystal Structure of Human CD1b with a Bound Bacterial Glycolipid
Authors: Batuwangala Thil , Shepherd Dawn , Gadola Stephan D. , Gibson Kevin J. C. , Zaccai Nathan R. , Fersht Alan R. , Besra Gurdyal S. , Cerundolo Vincenzo , Jones E. Yvonne
Published: April 2014

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains. The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.
Macrophages, CD4+ or CD8+ Cells Are Each Sufficient for Protection against Chlamydia pneumoniae Infection through their Ability to Secrete IFN-γ
Authors: Rothfuchs Antonio Gigliotti , Kreuger Maria Regina , Wigzell Hans , Rottenberg Martin E.
Published: April 2014

By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1−/−/common cytokine receptor γ-chain (γCR)), and T and B cell and IFN-γ-deficient (RAG-1−/−/IFN-γ−/−) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-γ secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-γ-dependent innate immune protection. Inoculation of wild type, but not IFN-γ−/− bone marrow-derived macrophages protected RAG-1−/−/IFN-γ−/− mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1−/− C. pneumoniae-infected mice expressed IFN-γ mRNA. Reconstitution of RAG-1−/−/γcR−/− or RAG-1−/−/IFN-γ−/− mice with CD4+ or CD8+ cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-γ−/− CD4+ or IFN-γ−/− CD8+ SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-γR−/− CD4+ or IFN-γR−/− CD8+ SC were inoculated into RAG-1−/−/IFN-γ−/− mice. Thus both nonlymphoid and T cell-derived IFN-γ can play a central and complementary role in protection against C. pneumoniae. IFN-γ secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-γ regulated T cell protective functions.
Recognition of Homo- and Heterosubtypic Variants of Influenza A Viruses by Human CD8+ T Lymphocytes
Authors: Boon Adrianus C. M. , de Mutsert Gerrie , van Baarle Debbie , Smith Derek J. , Lapedes Alan S. , Fouchier Ron A. M. , Sintnicolaas Kees , Osterhaus Albert D. M. E. , Rimmelzwaan Guus F.
Published: April 2014

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8+ CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP418–426), were generated. As determined in 51Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8+ cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.
Protective Immunization against Group B Meningococci Using Anti-Idiotypic Mimics of the Capsular Polysaccharide
Authors: Beninati Concetta , Arseni Simona , Mancuso Giuseppe , Magliani Walter , Conti Stefania , Midiri Angelina , Biondo Carmelo , Polonelli Luciano , Teti Giuseppe
Published: April 2014

Use of the serogroup B meningococcal capsular polysaccharide (MenB CP) as a vaccine is hampered by the presence of epitopes that cross-react with human polysialic acid. As non-cross-reactive, protective capsular epitopes have also been described, we set out to develop protein mimics of one of such epitopes using as a template a highly protective mAb (mAb Seam 3) raised against a chemically modified form of the MenB CP (N-Pr MenB CP). Using phage display, anti-idiotypic single-chain Ab fragments (scFvs) were obtained from spleen cells of mice immunized with the Seam 3 mAb. Two Seam 3-specific scFvs competed with N-Pr MenB CP for binding to either mAb Seam 3 or rabbit Abs present in typing sera. Moreover, in mice and rabbits the scFvs elicited the production of Abs reacting with both N-Pr MenB CP and whole meningococci, but not with human polysialic acid. These scFv-induced Ab responses were boostable and of the Th1 type, as shown by a predominance of IgG2a. In addition, passive immunization with sera from scFv-immunized animals partially protected neonatal mice from experimental infection with group B meningococci. In conclusion, we have produced anti-idiotypic scFvs that mimic a protective MenB CP epitope and may be useful in the development of an alternative group B meningococcal vaccine.
Cyclooxygenase-2-Mediated Prostaglandin E2 Production in Mesenteric Lymph Nodes and in Cultured Macrophages and Dendritic Cells after Infection with Salmonella
Authors: Bowman Christal C. , Bost Kenneth L.
Published: April 2014

Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE2 synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE2 production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE2 response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE2 secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE2 synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE2 synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type Salmonella.
HIV-1-Specific Memory CD4+ T Cells Are Phenotypically Less Mature Than Cytomegalovirus-Specific Memory CD4+ T Cells
Authors: Yue Feng Yun , Kovacs Colin M. , Dimayuga Rowena C. , Parks Paul , Ostrowski Mario A.
Published: April 2014

HIV-1-specific CD4+ T cells are qualitatively dysfunctional in the majority of HIV-1-infected individuals and are thus unable to effectively control viral replication. The current study extensively details the maturational phenotype of memory CD4+ T cells directed against HIV-1 and CMV. We find that HIV-1-specific CD4+ T cells are skewed to an early central memory phenotype, whereas CMV-specific CD4+ T cells generally display a late effector memory phenotype. These differences hold true for both IFN-γ- and IL-2-producing virus-specific CD4+ T cells, are present during all disease stages, and persist even after highly active antiretroviral therapy (HAART). In addition, after HAART, HIV-1-specific CD4+ T cells are enriched for CD27+CD28−-expressing cells, a rare phenotype, reflecting an early intermediate stage of differentiation. We found no correlation between differentiation phenotype of HIV-1-specific CD4+ T cells and HIV-1 plasma viral load or HIV-1 disease progression. Surprisingly, HIV-1 viral load affected the maturational phenotype of CMV-specific CD4+ T cells toward an earlier, less-differentiated state. In summary, our data indicate that the maturational state of HIV-1-specific CD4+ T cells cannot be a sole explanation for loss of containment of HIV-1. However, HIV-1 replication can affect the phenotype of CD4+ T cells of other specificities, which might adversely affect their ability to control those pathogens. The role for HIV-1-specific CD4+ T cells expressing CD27+CD28− after HAART remains to be determined.
Liver Dendritic Cells Present Bacterial Antigens and Produce Cytokines upon Salmonella Encounter
Authors: Johansson Cecilia , Wick Mary Jo
Published: April 2014

The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c+ cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8α−CD4− double negative cells constituted the majority of liver CD11c+ (∼85%) with few cells expressing CD8α or CD4. Flow cytometry analysis of freshly isolated CD11c+ cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c+ MHC class IIhigh cells had a greater capacity to internalize Salmonella relative to CD11c+ MHC class IIlow cells. Moreover, both CD8α− and CD8α+ liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c+ cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c+ cells infected with Salmonella expressing GFP showed that both CD8α− and CD8α+ DC produced IL-12p40 and TNF-α. The majority of cytokine-positive cells did not contain bacteria (GFP−) whereas only a minor fraction of cytokine-positive cells were GFP+. Furthermore, only ∼30–50% of liver DC containing bacteria (GFP+) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4+ and CD8+ T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.
Suppression of Osteoprotegerin Expression by Prostaglandin E2 Is Crucially Involved in Lipopolysaccharide-Induced Osteoclast Formation
Authors: Suda Koji , Udagawa Nobuyuki , Sato Nobuaki , Takami Masamichi , Itoh Kanami , Woo Je-Tae , Takahashi Naoyuki , Nagai Kazuo
Published: April 2014

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE2 production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE2 in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-κB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE2 is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE2 production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE2 production.
Role of Bradykinin B2 and B1 Receptors in the Local, Remote, and Systemic Inflammatory Responses That Follow Intestinal Ischemia and Reperfusion Injury
Authors: Souza Danielle G. , Lomez Eliane S. L. , Pinho Vanessa , Pesquero João Bosco , Bader Michael , Pesquero Jorge Luís , Teixeira Mauro M.
Published: April 2014

The administration of bradykinin may attenuate ischemia and reperfusion (I/R) injury by acting on B2Rs. Blockade of B2R has also been shown to ameliorate lesions associated with I/R injury. In an attempt to explain these contradictory results, the objective of the present work was to investigate the role of and interaction between B1 and B2 receptors in a model of intestinal I/R injury in mice. The bradykinin B2R antagonist (HOE 140) inhibited reperfusion-induced inflammatory tissue injury and delayed lethality. After I/R, there was an increase in the expression of B1R mRNA that was prevented by HOE 140. In mice that were deficient in B1Rs (B1R−/− mice), inflammatory tissue injury was abrogated, and lethality was delayed and partially prevented. Pretreatment with HOE 140 reversed the protective anti-inflammatory and antilethality effects provided by the B1R−/− phenotype. Thus, B2Rs are a major driving force for B1R activation and consequent induction of inflammatory injury and lethality. In contrast, activation of B2Rs may prevent exacerbated tissue injury and lethality, an effect unmasked in B1R−/− mice and likely dependent on the vasodilatory actions of B2Rs. Blockade of B1Rs could be a more effective strategy than B2 or B1/B2 receptor blockade for the treatment of the inflammatory injuries that follow I/R.
Contribution of Antigen-Primed CD8+ T Cells to the Development of Airway Hyperresponsiveness and Inflammation Is Associated with IL-13
Authors: Miyahara Nobuaki , Takeda Katsuyuki , Kodama Taku , Joetham Anthony , Taube Christian , Park Jung-Won , Miyahara Satoko , Balhorn Annette , Dakhama Azzeddine , Gelfand Erwin W.
Published: April 2014

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8+ T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8+ T cells in the development of these allergen-induced responses. CD8-deficient (CD8−/−) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8−/− mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8−/− mice with allergen-sensitized CD8+ T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8+ T cells or allergen-sensitized CD8+ T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8+ T cells were a source of IL-13. These data suggest that Ag-primed CD8+ T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.
Inflammatory Gene Profiles in Gastric Mucosa during Helicobacter pylori Infection in Humans
Authors: Wen Sicheng , Felley Christian P. , Bouzourene Hanifa , Reimers Mark , Michetti Pierre , Pan-Hammarström Qiang
Published: April 2014

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation. Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients. cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H. pylori-infected patients. Two main gene expression profiles were identified based on cluster analysis. The data obtained suggest a strong involvement of selected Toll-like receptors, adhesion molecules, chemokines, and ILs in the mucosal response. This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H. pylori. The presence of a “Helicobacter-infection signature,” i.e., a set of genes that are up-regulated in biopsies from H. pylori-infected patients, could be derived from this analysis. The genotype of the bacteria (presence of genes encoding cytotoxin-associated Ag, vacuolating cytotoxin, and blood group Ag-binding adhesin) was analyzed by PCR and shown to be associated with differential expression of a subset of genes, but not the general gene expression pattern. The expression data of the array hybridization was confirmed by quantitative real-time PCR assays. Future studies may help identify gene expression patterns predictive of complications of the infection.