bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen receptor‐induced apoptosis of human germinal center B cells is targeted to a centrocytic subset
Authors: Billian Gisèle , Mondière Paul , Berard Marion , Bella Chantal , Defrance Thierry
Published: March 2007

The outcome of the signals transduced through the B cell antigen receptor (BCR) depends both on their maturational stage and on the extent of receptor cross‐linking. It is established that the BCR‐mediated apoptosis of immature B cells represents an important mechanism for tolerance induction in the pre‐immune B cell compartment. We show here that mature germinal center (GC) B cells can re‐acquire sensitivity to BCR‐induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor‐triggered apoptosis upon CD40 stimulation, suggesting that this phenomenon may play a role in the shaping of the mature B cell repertoire in GC. Our data reveal that the death signal evoked through the BCR does not involve the Fcγ receptors, does not operate through the Fas/Fas ligand system, and can be blocked by interleukin‐4. Finally, we found that the acquisition of sensitivity to the death‐promoting effect of anti‐Ig antibodies in CD40‐stimulated GC B cell cultures correlates with the induction of a centrocytic phenotype. We propose that negative regulatory signals via the BCR may delete somatically mutated centrocytes with self‐reactivity.
MRL/lpr CD4−CD8− and CD8+ T cells, respectively, mediate Fas‐dependent and perforin cytotoxic pathways
Authors: Benihoud Karim , Bonardelle Danielle , Bobé Pierre , Kiger Nicole
Published: March 2007

Autoimmune‐prone MRL/lpr mice, homozygous for the lpr mutation, exhibit defective apoptosis and develop generalized lymphoproliferation with the accumulation of a double‐negative (DN: CD4−CD8−) T cell population. The capacity of lpr T lymphocytes to effectuate Fas‐ and perforin‐mediated cytotoxicity was investigated. Spleen and lymph nodes cells spontaneously lyse Fas+ targets (thymocytes) through a Fas‐mediated mechanism as a consequence of their overex‐pression of Fas ligand (FasL) confirmed by semiquantitative reverse transcription (RT)‐PCR and immunoprecipitation analysis. This cytotoxicity was greatly increased after stimulation of the effectors by phorbol myristate acetate (PMA)+ionomycin. Under these conditions, MRL/lpr spleen and LN cells exhibited strong Fas‐mediated Ca2+‐independent cytotoxic activity against wild‐type Fas+ (H‐2 compatible or incompatible) thymocytes or lipopolysaccharide (LPS)‐transformed blast cells. Such Fas‐mediated cytotoxic activity was also observed with C57BL/6‐lpr, but never with wild‐type C57BL/6 or MLR+/+ effectors. Depletion experiments showed that the effector cells of this Fas‐mediated cytotoxicity were DN T cells. This subset, which represent in vivo activated T cells, can spontaneously lyse Fas+ targets by a mechanism that does not need the interaction of the T cell receptor (TCR) with major histocompatibility complex molecule plus antigen. This lytic potential is increased by PMA+ionomycin, which sends a second activation signal to these primed T cells. Therefore, the small amounts of Fas receptor expressed on MRL/lpr tissues may account for their nonspecific autoimmune attack by DN cells. In Con A‐containing medium, which allows detection of the perforin‐mediated pathway against Fas− targets, cytotoxic CD8+ effectors were detected that are able to kill lpr thymocytes via a Ca2+‐dependent pathway. Thus, in MRL/lpr mice, these CD8+ cells could constitute potent cytotoxic effectors against cells presenting antigen to their TCR.
Differentiation of human dendritic cells from monocytes in vitro
Authors: Chapuis Françoise , Rosenzwajg Michelle , Yagello Micael , Ekman Marianne , Biberfeld Peter , Gluckman Jean Claude
Published: March 2007

Since either macrophages (Mϕ) or dendritic cells (DC) differentiate from monocytes (MO) depending on culture conditions, we investigated the relationship of the DC and Mϕ differentiation pathways. Culturing MO‐enriched blood mononuclear cells with Mϕ colony‐stimulating factor (M‐CSF) or with granulocyte/Mϕ (GM)‐CSF induced Mϕ with a different morphology and CD14/CD1a expression. In contrast, in cultures with GM‐CSF and interleukin (IL)‐4, cells rapidly became nonadherent and acquired DC morphology, ultrastructure, CD1a expression, and most DC markers; they lost membrane CD14 and CD64 and capacity of phagocytosis, displayed less CD68 than Mϕ, but retained nonspecific esterase activity. These DC directly developed from MO without proliferation inasmuch as only day 0 FACS‐sorted MO, but not small CD14− cells, differentiated into DC when cultured with GM‐CSF and IL‐4, or to Mϕ with M‐CSF. While overall cell numbers declined, DC numbers plateaued from culture day 2 onwards, indicating that most had differentiasted by then. This differentiation was radioresistant and occurred without [3H]thymidine incorporation. Commitment to differentiate into DC with GM‐CSF and IL‐4 was irreversible by day 2, since discontinuing IL‐4 at this point did not revert cells to Mϕ. Alternatively, cells rapidly converted to DC when IL‐4 was added from day 2 to cultures initiated with GM‐CSF only. If cultures were initiated with M‐CSF and switched to GM‐CSF and IL‐4 after 2 or 5 days, about half of the cells still converted to DC. Thus, the capacity of MO and even of Mϕ to differentiate into DC was conserved for at least this period. The increased capacity to stimulate the mixed leukocyte reaction correlated with the relative number of CD1a− cells at any time and under each condition tested, a confirmation that these cells functionally qualify as DC. Thus, MO and even Mϕ can be directed to differentiate into DC depending on the cytokine microenvironment.
Reduced susceptibility to Fas‐mediated apoptosis in B‐1 cells
Authors: Masuda Keiji , Wang Jiyang , Watanabe Takeshi
Published: March 2007

Elimination of activated T and B cells by Fas‐dependent apoptosis may contribute to the maintenance of peripheral tolerance. CD40 ligation was recently shown to up‐regulate Fas expression and enhance susceptibility to Fas‐mediated apoptosis in mouse splenic B cells. In the present study, we have investigated the regulation of Fas expression and Fas‐triggered apoptotis in mouse peritoneal B‐1 cells. B‐1 cells expressed a similar level of CD40 as that on B‐2 cells, and proliferated in response to a soluble CD40 ligand (CD40L)‐CD8α chimeric protein, suggesting that CD40 on B‐1 cells is functional. In contrast to B‐2 cells, B‐1 cells expressed Fas at only low levels in response to CD40L‐CD8α alone or CD40L‐CD8α+interleukin‐4, and were resistant to Fas‐mediated apoptosis following these treatments. While Fas expression could be induced in B‐1 cells to a comparable level as that in B‐2 cells by cross‐linking CD40L‐CD8α with an anti‐CD8α antibody, the sensitivity to Fas‐mediated apoptosis in B‐1 cells was significantly reduced compared with B2 cells. These results suggest that peritoneal B‐1 cells from normal mice have a lower susceptibility to Fas‐mediated apoptosis and may distinguish B‐1 from B‐2 cells. Similarly, B‐1 cells from the peritoneal cavity and spleen of autoimmune‐prone NZB mice exhibited reduced susceptibility to Fas‐mediated apoptosis relative to their B‐2 counterparts. NZB splenic B‐1 cells, however, were more susceptible to Fas‐mediated apoptosis than NZB peritoneal B‐1 cells. The results presented here raise the possibility that the reduced susceptibility to Fas‐triggered apoptosis in B‐1 cells might be an accelerating factor for the autoantibody production in NZB mice.
The generation of memory antigen‐specific cytotoxic T cell responses by CD28/CD80 interactions in the absence of antigen
Authors: Flynn Kirsten , Müllbacher Arno
Published: March 2007

The interaction of co‐stimulatory molecules CD80/CD86 on antigen‐presenting cells with CD28 on naive CD8+ cytotoxic T (Tc) cells is understood to be critical in the induction of Tc effectors. CD80 is capable of providing signal 2 for the activation of Tc cells, but has no effect if encountered in the absence of specific peptide/MHC complexes (signal 1). We have found that CD80 presented in vitro to resting memory viral‐immune or alloimmune Tc cells can provide sufficient stimulus for the generation of effector Tc cells in the absence of specific antigen, the peptide/MHC class I complex. Effector Tc cells generated in vitro from influenza‐ or class I alloantigen‐primed mice by co‐stimulation in the absence of antigen require exogenous interleukin (IL)‐2 signaling via the cell surface‐expressed IL‐2 receptor or, under conditions of IL‐2 blockade, exogenous IL‐7. Activation of memory Tc cells by signal 1 and 2 is independent of IL‐2 and IL‐7. Although memory influenza‐immune Tc cells did respond to CD80 in the absence of antigen, the presence of antigen +CD80 enabled an earlier induction of these Tc cells and they retained their lytic activity in vitro over a longer time period. The capacity of memory Tc cells to be activated by signal 2 alone provides one explanation for the observed heterogeneity of phenotype of memory T cells in vivo and a possible mechanism for the maintenence of memory in the absence of persisting antigen.
Differential contribution of Lck and Fyn protein tyrosine kinases to intraepithelial lymphocyte development
Authors: Page Stephanie T. , Van Oers Nicolai S. C. , Perlmutter Roger M. , Weiss Arthur , Pullen Ann M.
Published: March 2007

The developmental stages and the role of protein tyrosine kinases (PTK) in the maturation of CD3+CD8αα+ intraepithelial lymphocytes (IEL) have not been extensively characterized. However, comparisons of thymic and extrathymic T cell development indicate that these processes involve some distinct signaling and selection events. We used mice deficient in Lck, Fyn, or both Lck and Fyn to analyze the role that these src‐family PTK play in IEL development. In contrast to thymocyte development, we found that all IEL subsets develop in mice deficient for either kinase alone. However, lck‐/‐ animals exhibited reduced numbers of TcRαβ+CD8αα+ IEL, indicating that Lck is important in the development of these cells. Mice which lack both Lck and Fyn fail to generate TcRαβ+ IEL, suggesting that signaling through the preTcR, mediated by Lck and, to a lesser extent Fyn, is required for maturation of all TcRαβ+ IEL lineages. Interestingly, a small population of TcRγδ+CD8αα+ cells are apparent in lck‐/‐fyn‐/‐ animals, demonstrating that TcRαβ+CD8αα+ and TcRγδ+CD8αα+ IEL have distinct PTK requirements for their development or expansion. CD3−‐CD8α−CD44+ and CD3−CD8αα+CD16/32+B220+ cells comprise the majority of IEL in both lck‐/‐fyn‐/‐ and rag‐/‐ mice, while they are poorly represented in wild‐type controls. Comparison of the cell surface phenotype of these putative precursor IEL in lck‐/‐fyn‐/‐ and rag‐/‐ animals suggests that IEL maturation in these animals is arrested at an equivalent developmental stage. Overall, the data presented demonstrate that signals mediated by Lck or Fyn direct TcRαβ+CD8αα+ IEL maturation but are dispensable for the development of TcRγδ+CD8αα+ IEL.
B7‐1‐dependent co‐stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells
Authors: Deeths Matthew J. , Mescher Matthew F.
Published: March 2007

To characterize better the co‐stimulatory activity of native B7‐1 in the absence of other receptor/ligand interactions that might contribute to the response, B7‐1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7‐1 with anti‐T cell receptor (TCR) mAb on cell‐sized latex microspheres provided an effective stimulus for activation of both CD4+ and CD8+ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4+ T cell response was prolonged and resulted in efficient interleukin‐2 production and clonal expansion. In contrast, CD8+ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7‐1 mediated co‐stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7‐1 co‐stimulation is not sufficient to sustain helper‐independent CD8+ CTL responses. When the dose responses of CD4+ and CD8+ T cells to B7‐1 were compared, CD8+ T cells were found to require higher densities of B7‐1 to attain an equivalent level of activation, suggesting that the level of expression of B7‐1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7‐1 transfectants, purified B7‐1 did not provide co‐stimulation when presented on a surface separate from the TCR stimulus.
Interferon‐γ and interleukin‐4 regulate T cell interleukin‐12 responsiveness through the differential modulation of high‐affinity interleukin‐12 receptor expression
Authors: Gollob Jared A. , Kawasaki Hiroshi , Ritz Jerome
Published: March 2007

Interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL‐12‐induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN‐γ in response to IL‐12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL‐4 exhibit a greatly diminished response to IL‐12, whereas the IL‐12 response of T cells activated with PHA plus IFN‐γ is enhanced. Radiolabeled IL‐12 binding studies demonstrate that the impairment of T cell IL‐12 responsiveness by IL‐4 is associated with the down‐regulation of high‐affinity IL‐12 receptor expression. In contrast, the enhancement of IL‐12 responsiveness by IFN‐γ is associated with the up‐regulation of high‐affinity IL‐12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low‐affinity IL‐12 receptor β subunit (IL‐12Rβ), we show that neither IL‐4 nor IFN‐γ affect the expression of IL‐12Rβ, which we determine to be one of at least two low‐affinity subunits required for high‐affinity IL‐12 binding. These findings suggest that IL‐4 and IFN‐γ exert opposite effects on T cell IL‐12 responsiveness by differentially modulating the expression of low‐affinity IL‐12 receptor subunits that are distinct from IL‐12Rβ and required, together with IL‐12Rβ, for high‐affinity IL‐12 binding and IL‐12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.
Humoral immune response to the capsid components of recombinant adenoviruses: Routes of immunization modulate virus‐induced Ig subclass shifts
Authors: Gahéry‐Ségard Hanne , Juillard Véronique , Gaston Jesintha , Lengagne Renée , Pavirani Andrea , Boulanger Pierre , Guillet Jean‐Gérard
Published: March 2007

This study examines in detail the capsid‐specific humoral immune response of BALB/c mice after one single injection of a replication‐defective adenovirus. Two routes of immunization, intravenous (i.v.) and intraperitoneal (i.p.), were compared for the response induced against the adenovirus particle and the three major components of the viral capsid, hexon, penton base, and fiber. A single immunization with the replication‐defective adenovirus induces a long and persistent humoral response specific for the virus. However, the molecular components of the viral capsid are differentially recognized depending on the route of immunization. The sera from mice immunized i.p. recognized only the hexon protein and a preferential switch to the IgG2a subclass was obtained which remained stable 100 days post‐immunization. The sera obtained from mice immunized i.v. gave a more complex response. At the beginning of the response, an isotype bias toward the IgG2a subclass was observed, but the isotype distribution changed during the whole period of the response. Neutralizing activity was maximum 45 days after immunization by both routes, and no activity was detectable after 3 months. However, the i.v. serum displayed a higher neutralizing activity than the i.p. serum. The IgM antiviral antibodies appeared to be an important component of the neutralizing activity, and the two routes of immunization do not induce the same IgG isotypes to neutralize viral infectivity. Extension of these findings to human gene therapy using recombinant adenoviruses may help to characterize the precise viral protein targets of neutralizing antibodies.
Differential antigen recognition by T cell populations from strains of mice developing polar forms of granulomatous inflammation in response to eggs of Schistosoma mansoni
Authors: Hernandez Hector J. , Trzyna Wendy C. , Cordingley John S. , Brodeur Peter H. , Stadecker Miguel J.
Published: March 2007

In humans, infection with schistosome helminths can lead to dissimilar forms of clinical disease. Likewise, in the experimental mouse system, identical infection protocols with Schistosoma mansoni cause a more severe granulomatous disease in the C3H strain than in the C57BL/6 strain. To address this difference, we developed panels of schistosomal egg antigen (SEA)‐specific T cell hybridomas to compare the responses of C3H and C57BL/6 mice to the major egg antigen p40. All derived C3H T cell hybridomas, despite being clonally distinct and restricted by either I‐Ak or I‐Ek, responded to recombinant fragment 15‐1 of the p40 antigen, while none of the C57BL/6 T cell hybridomas did. Consistent with the observed monoclonal T cell responses, polyclonal lymph node cells from schistosome‐infected C3H mice reacted strongly to fragment 15‐1, which contrasted sharply with the weak response displayed by the C57BL/6 strain. Moreover, studies with congenic mice demonstrated that the strong CD4+ T cell response to fragment 15‐1 was under major histocompatibility complex control and segregated with the H‐2k haplotype. These findings suggest that a dominant T cell response against a major egg antigen may represent a risk factor for the development of severe disease.
Altered Th1/Th2 balance associated with non‐major histocompatibility complex genes in collagen‐induced arthritis in resistant and non‐resistant rat strains
Authors: Müssener Åsa , Lorentzen Johnny C. , Kleinau Sandra , Klareskog Lars
Published: March 2007

Collagen‐induced arthritis (CIA) is a T cell‐dependent disease in which susceptibility is controlled by genes both within and outside the major histocompatibility complex (MHC). In the present study, we compared the humoral responses and kinetics of cytokine secretion patterns in the draining lymph nodes of arthritis‐susceptible DA rats and arthritis‐resistant F344 and DA MHC congenic PVG.1AV1 rats immunized with rat type II collagen (RCII) in incomplete Freund's adjuvant. The results demonstrate a marked humoral RCII response and a Th1 cytokine profile, with expression of interferon‐γ and interleukin (IL)‐2 mRNA in DA rats; a limited humoral RCII response and a Th2 cytokine profile, with expression of IL‐4 mRNA in arthritis‐resistant F344 rats; and a marked humoral RCII response in arthritis‐resistant PVG.1AV1 rats. However, in contrast to DA rats, PVG.1AV1 rats produce IgG1 autoantibodies which, together with strong expression of IL‐4 mRNA, indicates the involvement of Th2 subsets. From these data, we conclude that non‐MHC gene(s) determines the direction of the anti‐RCII response towards a Th1 disease‐promoting, or a Th2 disease‐limiting response.
Transgenic expression of a CD46 (membrane cofactor protein) minigene: Studies of xenotransplantation and measles virus infection
Authors: Thorley Bruce R. , Milland Julie , Christiansen Dale , Lanteri Marc B. , McInnes Beth , Moeller Ingrid , Rivailler Pierre , Horvat Branka , Rabourdin‐Combe Chantal , Gerlier Denis , McKenzie Ian F. C. , Loveland Bruc. E. E.
Published: March 2007

CD46 (membrane cofactor protein) is a human cell‐surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody‐dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high‐level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.
Pax‐5 is identical to EBB‐1/KLP and binds to the VpreB and λ5 promoters as well as the KI and KII sites upstream of the Jχ genes
Authors: Tian Jiayuan , Okabe Taijiro , Miyazaki Takahiro , Takeshita Sunao , Kudo Akira
Published: March 2007

During B cell differentiation, the pre‐B cell stage plays a significant role in immunoglobulin gene rearrangement in the context of the allelic exclusion and χ chain gene rearrangement. We previously reported that the early B cell‐specific binding protein (EBB)‐1 transcription factor binds to the promoters of two pre‐B cell‐specific genes, VpreB and λ5, and regulates their pre‐B cell‐specific expression. Here, we demonstrate that EBB‐1 binds to the KI and KII sites in the upstream of Jχ region, which are crucial for χ chain gene rearrangement. Gene transfer and gel‐shift assays demonstrate that EBB‐1 is identical to Pax‐5 and binds to promoters of VpreB and λ5 as well as the KI and KII sites. Our results suggest that Pax‐5 plays an important role in the coordinate regulation of several immunoglobulin gene family members that are crucial in B cell development.
The T cell receptor β genes of Xenopus
Authors: Chrétien Isabelle , Marcuz Anne , Fellah Julien , Charlemagne Jacques , Pasquier Louis Du
Published: March 2007

cDNA of the T cell receptor β (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. Ten BV families, two D segments, ten J segments, and a single C region have been defined so far. Each V family consists of one to two members per haploid genome. A unique feature of the Xenopus TCRB constant region is the lack of N‐linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is inherited in a diploid manner despite the pseudotetraploidy of the Xenopus laevis and X. gilli used in this study.
Carboxy‐terminal residues of major histocompatibility complex class II‐associated peptides control the presentation of the bacterial superantigen toxic shock syndrome toxin‐1 to T cells
Authors: Wen Renren , Broussard Dana R. , Surman Sherri , Hogg Twala L. , Blackman Marcia A. , Woodland David L.
Published: March 2007

Previous studies have shown that the presentation of some bacterial superantigens by major histocompatibility complex (MHC) class II molecules is strongly influenced by class II‐associated peptides. For example, presentation of the toxic shock syndrome toxin‐1 (TSST‐1) superantigen by antigen‐processing‐defective T2‐I‐Ab cells (which expresses I‐Ab that is either empty or associated with invariant chain‐derived peptides) can be strongly enhanced by some, but not other, I‐Ab‐binding peptides. Here we investigate the contribution of I‐Ab‐associated peptides in the presentation of TSST‐1 to T cells. The data show that overlapping peptides expressing the same core I‐Ab‐restricted epitope, but with various N and C termini, can differ profoundly in their ability to promote TSST‐1 presentation to T cells. Analysis of altered and truncated peptides indicates that residues at the C‐terminal end of the peptide have a dramatic effect on TSST‐1 presentation. This effect does not involve a cognate interaction between the peptide and the TSST‐1 molecule, but appears to depend on the length of the C‐terminal region. These data are consistent with crystallographic studies suggesting that TSST‐1 may interact with the C‐terminal residues of MHC class II‐associated peptides. We also examined the capacity of naturally processed peptides to promote TSST‐1 binding using a superantigen blocking assay. The data demonstrated that a naturally processed epitope is dominated by peptides that do not promote strong TSST‐1 binding to I‐Ab. Taken together, these data suggest that TSST‐1 binding to MHC class II molecules is controlled by the C‐terminal residues of the associated peptide, and that many naturally processed peptide/class II complexes do not present TSST‐1 to T cells. Thus, the peptide dependence of TSST‐1 binding to class II molecules may significantly reduce the capacity of TSST‐1 to stimulate T cells.
Superantigen and endotoxin synergize in the induction of lethal shock
Authors: Blank Christian , Luz Arne , Bendigs Sylvia , Erdmann Andreas , Wagner Hermann , Heeg Klaus
Published: March 2007

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro‐inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100‐fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin‐6, and interferon‐γ (IFN‐γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D‐galactosamine (D‐GalN). Opposed to D‐GalN‐pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti‐IFN‐γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell‐derived IFN‐γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB‐induced T cell deletion and anergy induction. Since Gram‐positive and Gram‐negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.
Naive alloreactive CD8 T cells are activated by purified major histocompatibility complex class I and antigenic peptide
Authors: Goldstein Julia , Mostowsky Howard , Tung Joyce , Hon Huiming , Brunswick Mark , Kozlowski Steven
Published: March 2007

In this report, we demonstrate stimulation of T cell receptor (TCR) transgenic CD8 T cells by isolated major histocompatibility complex (MHC) class I H‐2Ld complexes and antigenic peptide. This is the first demonstration of CD8 T cells activated by MHC and antigenic peptide in the absence of antigen priming. Furthermore, isolated MHC and a potent peptide antigen can stimulate phenotypically naive CD44− T cells to become CTL effectors and to produce interleukin‐2 in nanogram per milliliter amounts. These results demonstrate that particular TCR antigen pairs may overcome the need for specialized antigen‐presenting cells and have implications for mechanisms of autoimmunity and tolerance induction.
Surfactant protein A, but not surfactant protein D, is an opsonin for influenza A virus phagocytosis by rat alveolar macrophages
Authors: Benne Cornelis A. , Benaissa‐Trouw Barry , Van Strijp Jos A. G. , Kraaijeveld Cornelis A. , Van Iwaarden J. Freek F.
Published: March 2007

Surfactant protein A (SP‐A) and surfactant protein D (SP‐D) are collectins, and both proteins were shown to interact with influenza A virus and alveolar macrophages. However, it is not known whether SP‐A and SP‐D can serve as opsonins for the phagocytosis of influenza A virus by alveolar macrophages. In the present study, we investigated the opsonic activities of SP‐A and SP‐D for phagocytosis of fluorescein isothiocyanate (FITC)‐labeled influenza A (H3N2) virus by rat alveolar macrophages using flow cytometry. SP‐A enhanced the association of the virus with macrophages in a dose‐dependent manner, reaching a maximum at an SP‐A concentration of 60 μg/ml. An approximate threefold increase in association of influenza A virus with alveolar macrophages in the presence of SP‐A over control incubations which contained no SP‐A was observed. Half of the total cell‐associated fluorescence could be quenched as demonstrated using the extracellular quenching dye trypan blue. These results indicate that SP‐A mediates internalization of FITC‐labeled influenza A (H3N2) virus by alveolar macrophages. Removal of the carbohydrate moiety of SP‐A by N‐glycosidase F treatment or cleavage of its sialic acid residues by neuraminidase abolished the enhancement of the phagocytosis of FITC‐labeled influenza A virus by alveolar macrophages. Mannan, a mannose homopolysaccharide known to bind to the carbohydrate‐binding domain of SP‐A, did not affect the SP‐A‐mediated phagocytosis of FITC‐labeled influenza by alveolar macrophages. In contrast, SP‐D neither enhanced the association of FITC‐labeled influenza A virus with alveolar macrophages nor affected the opsonic activity of SP‐A for FITC‐labeled influenza A (H3N2) virus at the SP‐D concentrations tested. It is concluded that SP‐A acts via its sialic acid residues as an opsonin in the phagocytosis of influenza A virus by alveolar macrophages.
Expression of two interleukin‐15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides
Authors: Meazza Raffaella , Gaggero Alessia , Neglia Francesca , Basso Stefania , Sforzini Sabrina , Pereno Raffaele , Azzarone Bruno , Ferrini Silvano
Published: March 2007

Interleukin (IL)‐15 is a four‐helix bundle cytokine sharing several biological properties with IL‐2. By reverse transcriptase‐polymerase chain reaction analysis, human cancer cell lines of different histotypes are shown to express two IL‐15 amplification products: a 524‐bp band corresponding to the IL‐15 mRNA found in macrophages, and another of 643 bp corresponding to an alternatively spliced mRNA including a 119‐bp alternative exon. IL‐15 was undetectable in the supernatant of tumor cell lines expressing either one or both of the mRNA isoforms as evaluated by a bioassay or by ELISA, indicating that IL‐15 is not secreted. However, IL‐15 could be detected intracellularly in some tumor cells by confocal microscopy analysis. Since the pre‐proteins encoded by the two mRNA isoforms differ in the signal peptide sequence, we have analyzed the characteristics of these signal peptides and their possible role in controlling secretion. The two IL‐15 cDNA isoforms, expressed in COS‐7 cells, induced very low levels of IL‐15 secretion. However, substitution of the sequence encoding natural signal peptide(s) with the one from IgVχ chain in the IL‐15 cDNA results in a significantly higher secretion of biologically active IL‐15 (15–30‐fold) upon cDNA transfection. A poor efficiency of natural signal peptides may represent one of the mechanisms involved in the control of IL‐15 secretion.
RANTES stimulation of T lymphocyte adhesion and activation: role for LFA‐1 and ICAM‐3
Authors: Szabo Margit C. , Butcher Eugene C. , McIntyre Bradley W. , Schall Thomas J. , Bacon Kevin B.
Published: March 2007

The chemokine RANTES is a potent chemoattractant and activator of T lymphocytes. Mechanisms underlying the RANTES‐induced activation of T lymphocytes leading to adhesion and migration have not been fully analyzed. We investigate here the function of RANTES in the regulation of T cell adhesion, specifically the induction of homotypic aggregation. RANTES induced the expression of many important cell surface adhesion and activation receptors in a normal human T cell clone and peripheral blood T lymphocytes, including members of the β1 and β2 integrin family, CD44, CD50, and CD28. Up‐regulation of these markers correlated with RANTES‐stimulated homotypic adhesion of T cells. This homotypic aggregation event was RANTES dose‐dependent, prolonged, and pertussis toxin‐independent, but herbimycin A‐sensitive, suggesting that it involves signaling through alternative (Gαi protein‐independent) pathways. Using specific monoclonal antibodies, the homotypic aggregation event was shown to be lymphocyte function‐associated antigen‐1 (LFA‐1)‐dependent, with no observable interaction through α4 or β1 integrins. Intercellular adhesion molecule‐3 (ICAM‐3) and possibly ICAM‐1 participate as LFA‐1 ligands. Additionally, RANTES phosphorylated the β chain of LFA‐1 1–2 min following stimulation. These results imply a specific role for the chemokine RANTES in T cell activation and intercellular adhesion.
Cytokine induction of monocyte chemoattractant protein‐1 gene expression in human endothelial cells depends on the cooperative action of NF‐χB and AP‐1
Authors: Martin Teresa , Cardarelli Pina M. , Parry Graham C. N. , Felts Katherine A. , Cobb Ronald R.
Published: March 2007

Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C‐C chemokine is monocyte chemoattractant protein‐1 (MCP‐1) which is synthesized by a variety of cell types including endothelial cells in response to a variety of stimuli. MCP‐1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine‐induced MCP‐1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)‐χB‐like binding site and the AP‐1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP‐1 promoter by interleukin‐(IL)‐1β. Site‐directed mutagenesis or deletion of the NF‐χB‐like site decreased the cytokine‐induced activity of the promoter. Site‐directed mutagenesis of the AP‐1 binding site also decreased the cytokine‐induced activity of the promoter. We show that the NF‐χB‐like site located at −90 in the MCP‐1 promoter binds to the p50/p65 heterodimer of the NF‐χB/Rel family in IL‐1β‐stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP‐1 promoter as well. The data presented in this study suggest that cytokine‐induced MCP‐1 gene expression in human endothelial cells depends on the cooperative action of NF‐χB and AP‐1.
Agonist antibody and Fas ligand mediate different sensitivity to death in the signaling pathways of Fas and cytoplasmic mutants
Authors: Thilenius Anja R. B. , Braun Kari , Russell John H.
Published: March 2007

We have produced three forms of human Fas: full‐length Fas, Fas with a C‐terminal deletion, and a chimera between extracellular Fas and the intracellular domain of the tumor necrosis factor receptor I p55 subunit. We transfected cell lines with these constructs to compare the relative capacity of antibody agonists and the physiological Fas ligand (FasL) to stimulate death. With two agonistic antibodies, the chimera is 100‐ to 1000‐fold more sensitive to induction of death than the full‐length Fas. The C‐terminal deletion mutant also shows greatly enhanced death in comparison to the wild‐type receptor. In contrast, when FasL is used to trigger the Fas pathway, wild‐type Fas and the deletion mutant are similarly sensitive, whereas the chimera is 100‐fold less susceptible to ligand‐mediated killing than Fas. This demonstrates that antibody agonists and natural ligand can stimulate different signaling pathways and emphasizes the limitations of defining physiologically important signaling pathways solely by antibody agonists.
The HER2/neu‐derived peptide p654–662 is a tumor‐associated antigen in human pancreatic cancer recognized by cytotoxic T lymphocytes
Authors: Peiper Matthias , Goedegebuure Peter S. , Linehan David C. , Ganguly Eric , Douville Cara C. , Eberlein Timothy J.
Published: March 2007

The protooncogene HER2/neu encodes a 185‐kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that cytotoxic T lymphocytes (CTL) derived from breast, ovarian, and non‐small cell lung cancer recognized a peptide derived from HER2/neu. To evaluate whether this HLA‐A2‐binding peptide is a tumor‐associated antigen (TAA) in pancreatic cancer, the ability of HER2/neu‐reactive CTL to lyse human pancreatic carcinoma cells was tested. CTL were generated from tumor‐associated T lymphocytes from HLA‐A2+ HER2/neu+ breast and ovarian cancer patients. All CTL recognized autologous and allogeneic HER2/neu+ tumor cells in an HLA‐A2‐restricted fashion. Furthermore, all CTL recognized p654–662 (GP2) derived from HER2/neu. These CTL also recognized HER2/neu+ pancreatic cancer cells in an HLA‐A2‐restricted fashion. HER2/neu+ HLA‐A2− pancreatic cancer cells were not or only poorly lysed. Repeated stimulation of HLA‐A2+ PBL from pancreatic cancer patients using the HER2/neu‐derived peptide resulted in specific recognition of this peptide and, more importantly, HER2/neu+ pancreatic tumors in an HLA‐A2‐restricted fashion. Autologous HLA‐A2+ fibroblasts or HLA‐A2+ malignant melanoma cells were not recognized. HLA‐A2− peptide‐stimulated T lymphocytes showed no significant cytotoxicity. These results demonstrate that this HER2/neu‐derived peptide is a shared TAA among several adenocarcinomas including pancreatic carcinoma, suggesting a common mechanism of recognition of these human tumors by T lymphocytes. The identification of the HER2/neu‐derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies.
Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus
Authors: Bommhardt Ursula , Cole Michael S. , Yun Tso J. , Zamoyska Rose
Published: March 2007

Differentiation of thymocytes into mature single‐positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), coreceptors (CD4 or CD8) and their appropriate major histocompatibility complex‐encoded ligands. Precisely how these receptor/co‐receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by coligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')2 antibodies to mimic TCR/co‐receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross‐linking or targeting to Fc receptor‐bearing cells. We show that TCR/CD3 co‐ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co‐ligation with CD8 also induces maturation of CD4T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co‐ligation of TCR and CD8 appear weaker than from co‐ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single‐positive cell differentiation.
Expression of class II, but not class I, major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni
Authors: Hernandez Hector J. , Wang Yong , Tzellas Nia , Stadecker Miguel J.
Published: March 2007

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4+ T helper lymphocytes sensitized to parasite egg antigens. However, CD8+ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4+ and CD8+ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4+ and CD8+ T cells. The results clearly show that schistosome‐infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild‐type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon‐gamma, interleukin (IL)‐4, IL‐10] responses; nor were they able to present egg antigens to specifically sensitized CD4+ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild‐type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II‐restricted CD4+ T helper cells. They also suggest that CD8+ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.
Molecular cloning of the mouse proteasome subunits MC14 and MECL‐1: reciprocally regulated tissue expression of interferon‐γ‐modulated proteasome subunits
Authors: Stohwasser Ralf , Standera Sybille , Peters Inga , Kloetzel Peter‐M. , Groettrup Marcus
Published: March 2007

The primary structures of the interferon‐γ‐inducible mouse 20S proteasome subunit MECL‐1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL‐1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in nonlymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL‐1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and δ as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells.
Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules
Authors: Anderson Graham , Anderson Kim L. , Tchilian Elma Z. , Owen John J. T. , Jenkinson Eric J.
Published: March 2007

We have investigated the role of specific components of the thymic stroma during development of CD4− 8− T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44− precursors, develop to the CD4+8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1‐ethyl‐3‐(3'‐dimethylaminopropyl)carbodiimide (ECDI)‐treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast‐associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to α4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast‐associated extracellular matrix.
Human interdigitating dendritic cells directly stimulate CD40‐activated naive B cells
Authors: Björck Pia , Flores‐Romo Leopoldo , Liu Yong‐Jun
Published: March 2007

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40+ lineage‐negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high‐level expression of major histocompatibility complex class II, the co‐stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL‐2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co‐localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.