bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial perturbations define lymphocytes undergoing apoptotic depletion in vivo
Authors: Castedo Maria , Macho Antonio , Zamzami Naoufal , Hirsch Tamara , Marchetti Philippe , Uriel José , Kroemer Guido
Published: March 2007

We have recently shown that lymphocyte apoptosis induced by dexamethasone or superantigens is accompanied by reduction of mitochondrial transmembrane potential (ΔΨm) which precedes nuclear DNA fragmentation. Here, we demonstrate that fluorochromes such as 3,3′dihexyloxacarbocyanine iodide [DiOC6(3)] which measure ΔΨm, or fluorochromes such as hydroethidine (HE) which measure mitochondrial superoxide anion production allow the identification of thymocytes or peripheral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic α/β T cell receptor (TCR) specific for a class I‐restricted male‐specific peptide, the superoxide‐mediated oxidation of HE into ethidium (Eth) is enhanced among thymocytes which are being deleted due to negative selection (CD4+ CD8+ cells expressing the transgenic TCR in male mice) or lack of positive selection (CD4+ CD8− thymocytes from female mice). ΔΨm reduction and/or enhanced HE oxidation are also found when apoptosis is induced by a series of pathogenic agents. Thus, lethal irradiation provokes mitochondrial and nuclear signs of apoptosis, and both these alterations are absent in mice bearing a p53 null mutation, underlying the correlation between mitochondrial perturbation and nuclear apoptosis. Similarly, superantigen‐triggered deletion of peripheral T cells in vivo is accompanied by enhanced HE → Eth conversion and reduced DiOC6(3) uptake. More importantly, as compared to normal controls. CD4+ or CD8+ cells from clinically asymptomatic human immunodeficiency virus‐1 (HIV‐1) carriers also contain a significantly elevated percentage of cells endowed with reduced DiOC6(3) uptake. In superantigen‐ and HIV‐induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC6(3) uptake is more important than that of cells marked by enhanced HE → Eth conversion. In conclusion, mitochondrial alterations precede and/or accompany nuclear signs of apoptosis induced by physiological and a variety of different pathogenic agents in vivo.
Altered Th1/Th2 balance associated with the immunosuppressive/protective effect of the H‐2Ab allele on the response to allo‐4‐hydroxyphenylpyruvate dioxygenase
Authors: Brunner Monika , Larsen Sanne , Sette Alessandro , Mitchison Avrion
Published: March 2007

The H‐2Ab allele exerts a dominant down‐regulatory effect on the anti‐allo‐HPPD (4‐hydroxyphenylpyruvate dioxygenase) antibody response, through a hitherto unknown mechanism. In the present study, the allo‐variable peptide bound to responder H‐2Ak molecules with higher affinity than to H‐2Ab ones, arguing against the operation of an affinity hierarchy. Quantitative polymerase chain reaction revealed differences in cytokine mRNA expression between suppressed and high‐responder mice. Lymph node cells of responder but not suppressed mice contained high levels of interleukin (IL)‐4 mRNA as early as 11 h post‐immunization and continued to do so for at least 8 days; this early burst was paralleled by a small burst in transforming growth factor (TGF)‐β mRNA level. Differences in IL‐12 mRNA were not detected, although an early IL‐12 effect could not be excluded. Interferon (IFN)‐γ appeared to contribute to the suppression at later time points. Early treatment of responder mice with anti‐IL‐4 monoclonal antibody (11B11) down‐regulated the antibody response. The proliferative T cell response from hyperimmunized mice was reduced but still detectable in the presence of an H‐2Ab allele. Thus, in the presence of this allele, the Th1 response is enhanced and that of Th2 cells suppressed, apparently as a result of the bias of H‐2Ab‐restricted T cells in favor of the Th1 subset.
Human T lymphocyte activation induces tyrosine phosphorylation of α‐tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase
Authors: Marie‐Cardine Anne , Kirchgessner Henning , Eckerskorn Christoph , Meuer Stefan C. , Schraven Burkhart
Published: March 2007

A glutathione‐S‐transferase‐src‐homology domain 2 (GST‐SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, α‐ and β‐tubulin were identified by N‐terminal sequencing. Further analysis established that α‐tubulin exists as a tyrosine‐phosphorylated protein in Jurkat cells, where it interacts with p59fyn, but not with p56lck. By contrast, in untransformed resting human T lymphocytes α‐tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine‐phosphorylated α‐tubulin that is able to interact with the fyn‐SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.
The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins
Authors: Bailly Pascal , Tontti Eveliina , Hermand Patricia , Cartron Jean‐Pierre , Gahmberg Carl G.
Published: March 2007

Leukocyte adhesion involves the leukocyte‐specific integrins CD11a/CD18, CD11b/CD18 and CD11c/CD18, which bind to intercellular adhesion molecules (ICAM). Three ICAM have been described, and are expressed on leukocytes and various other cells, but are absent from red cells. Here, we show that the red cell Landsteiner‐Wiener (LW) blood group glycoprotein is an ICAM which binds to the leukocyte‐specific integrins. This finding has important implications in red cell physiology.
Clustered and interspersed gene families in the mouse immunoglobulin ϰ locus
Authors: Zocher Ines , Röschenthaler Franz , Kirschbaum Thomas , Schäble Karlheinz F. , Hörlein Rita , Fleischmann Barbara , Kofler Reinhard , Geley Stephan , Hameister Horst , Zachau Hans G.
Published: March 2007

Although numerous solitary germ‐line Vϰ genes and two small Vϰ contiguously cloned gene regions (contigs) are known, no attempts to systematically elucidate the structure of the ϰ locus of the mouse have been reported so far. As a first step to this aim we screened a cosmid library of C57BL/6J mouse DNA with 18 probes that are more or less specific for the different Vϰ gene families. Ninety‐one Vϰ gene‐containing cosmid clones were characterized by detailed restriction mapping and hybridizations. Several contigs were constructed from overlapping clones. The contigs and the still unlinked cosmid clones cover 1.6 Mb. Many of the cosmid clones were localized on chromosome 6 where the ϰ locus is known to reside; no evidence for the existence of dispersed Vϰ genes (orphons) was obtained. Eighty‐five strong hybridization signals were assigned to distinct Vϰ gene families, while for 11 weak signals the assignment was less definite. As to the distribution of gene families within the locus the following situation emerged: there are both, groups of genes which belong to one Vϰ gene family (“clusters”) and groups in which genes of different families are interspersed. The interspersion of gene families seems to be more pronounced than has been assumed so far. Additional Vϰ genes which are known to exist will have to be isolated from other gene libraries of the same mouse Igϰ haplotype.
The role of perforin‐expression by granular metrial gland cells in pregnancy
Authors: Stallmach Thomas , Ehrenstein Tilman , Isenmann Stefan , Müller Christoph , Hengartner Hans , Kägi David
Published: March 2007

The pregnant uterus of humans and rodents contains a population of granulated lymphoid cells, which, in the mouse, are called granular metrial gland (GMG) cells and have been described to express high levels of perforin. Since there is evidence for cytolytic activity of these cells and since perforin is a crucial effector molecule for the lytic action of cytotoxic T cells and natural killer cells, we evaluated the function of perforin in the pregnant uterus by using perforin‐deficient mice. Perforin‐deficient female mice were found to reproduce as efficiently as normal control females when bred either with syngeneic or allogeneic males. However, perforin‐deficient mice differed from normal mice in that the frequency of GMG cells was significantly higher within maternal blood spaces and within several compartments of the feto‐maternal interface. Proliferating GMG cells, identified by [3H] thymidine incorporation, were observed during more advanced stages of pregnancy when compared to normal controls. In contrast to normal mice, perforin‐deficient mice did not display GMG cells attached to degenerating trophoblasts; instead perforin‐deficient GMG cells were often observed in association with small maternal lymphocytes. In addition, the lack of transmission of lymphocytic choriomeningitis virus from infected pregnant perforin‐deficient mice to the fetuses argued against a role of perforin expression by GMG cells in prevention of virus transmission from the mother to the fetus. Our data indicate that functional perforin is not necessary for successful pregnancies. The morphological changes in the pregnant uterus of perforin‐deficient mice might, however, point to a certain, as‐yet undefined function of perforin in the uterus of pregnant normal mice, which is functionally compensated in perforin‐deficient mice.
Detection of restricted isoform expression and tyrosine phosphatase activity of CD45 in murine dendritic cells
Authors: Haidl Ian D. , Ng David H. W. , Rothenberger Sylvia , Johnson Pauline , Jefferies Wilfred A.
Published: March 2007

CD45 is a cell surface transmembrane tyrosine phosphatase. It is expressed as distinct protein isoforms via alternative splicing of exons 4, 5 and 6. In T and B lymphocytes, CD45 is thought to play a critical role in antigen‐dependent signaling through their respective antigen receptor complexes. However, the isoform expression and enzymatic activity of CD45 in other leukocytes remains largely unknown. Here, we examine the isoform expression and phosphatase activity of CD45 in murine dendritic cells (DC). Flow cytometric double‐labeling analysis and biochemical analysis of purified splenic DC CD45 demonstrate that DC express both the CD45RB and CD45RO isoforms. Flow cytometric analyses of freshly isolated splenic DC and thymic DC also indicate the expression of CD45RB and CD45RO on these DC populations. In addition, we find that purified splenic DC CD45 possesses a high level of intrinsic tyrosine phosphatase activity. These data therefore establish the restricted isoform expression pattern of CD45 in murine DC and demonstrate that cells lacking specific antigen receptor complexes have active tyrosine phosphatase activity associated with CD45.
Presentation of endogenous viral proteins in association with major histocompatibility complex class II: On the role of intracellular compartmentalization, invariant chain and the TAP transporter system
Authors: Oxenius Annette , Bachmann Martin F. , Ashton‐Rickardt Philip G. , Tonegawa Susumu , Zinkernagel Rolf M. , Hengartner Hans
Published: March 2007

Major histocompatibility complex (MHC) class II‐associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously sythesized antigens. We have studied the MHC class II‐associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis. it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II‐associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non‐professional APC. We found that class II‐associated presentation of endogenously synthesized membrane associated LCMV‐GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II‐associated presentation of endogenously synthesized cytosolic LCMV‐NP was not observed even in Ii‐deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV‐GP apparently does not require processing in acidie endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading.
Differential requirements of CD45 for lymphocyte development and function
Authors: Kong Young‐Yun , Kishihara Kenji , Sumichika Hiroshi , Nakamura Takao , Kaneko Masahiro , Nomoto Kikuo
Published: March 2007

CD45 exon 6‐deficient mice show a profound block of thymocyte development at a transitional differentiation step from immature CD4+ CD8+ to mature single‐positive cells. Only a few T lymphocytes are observed in the periphery of such animals, but B cell development is not affected. We investigated whether mature B lymphocytes in CD45 exon 6‐deficient mice have normal functions in the absence of CD45 expression and show here that CD45‐defective B lymphocytes from CD45 exon 6‐deficient mice have intact B lymphocyte functions, including T‐dependent and T‐independent antigen‐specific antibody production and class switching in vivo if normal CD4+ T lymphocytes were adoptively transferred into the mutant mice. From these results, we conclude that CD45 expression on B lymphocytes is not essential for B cell responses following antigen stimulation in vivo. However, CD4+ T helper function was severely diminished in CD45 exon 6‐deficient mice, suggesting that the requirements of CD45 molecules in antigen‐specific response and development are different between T and B lymphocytes.
C‐C chemokines, but not the C‐X‐C chemokines interleukin‐8 and interferon‐γ inducible protein‐10, stimulate transendothelial chemotaxis of T lymphocytes
Authors: Roth Stephen J. , Carr Michelle Woldemar , Springer Timothy A.
Published: March 2007

Eight chemokines were tested for ability to elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes. The C‐C chemokines monocyte chemotactic protein (MCP)‐2, MCP‐3, RANTES, macrophage inflammatory protein (MCP)‐lα, MIP‐1β, and, as previously described, MCP‐1 induced significant, dose‐dependent transendothelial chemotaxis of CD3+ T lymphocytes. In contrast, the C‐X‐C chemokines interleukin‐8 (IL‐8) and interferon‐γ inducible protein‐10 (IP‐10) failed to induce transendothelial chemotaxis of CD3+ T lymphocytes or T lymphocyte subsets. RANTES, MIP‐1α, and MIP‐1β induced significant transendothelial chemotaxis of CD4+, CD8+, and CD45RO+ T lymphocyte subsets. Phenotyping of mononuclear cells that underwent transendothelial migration to MCP‐2, MCP‐3, RANTES, or MIP‐1α showed both monocytes and activated (CD26 high), memory‐type (CD45RO+) T cells. Both CD4+ and CD8+ T lymphocytes were recruited, but not natural killer cells or significant numbers of B cells. MCP‐2 was the only C‐C chemokine tested that attracted a significant number of naive‐type (CD45RA+) T lymphocytes. In the absence of endothelium, IL‐8 but not IP‐10 promoted modest but significant chemotoxis of CD3+ T lymphocytes. Our data support the hypothesis that C‐C, not the C‐X‐C chemokines IL‐8 or IP‐10, promote transendothelial chemotaxis of T lymphocytes.
Tolerance and immunity to the inducible self antigen C‐reactive protein in transgenic mice
Authors: Klein Thomas C. , Döffinger Rainer , Pepys Mark B. , Rüther Ulrich , Kyewski Bruno
Published: March 2007

The understanding of immunological tolerance has been greatly aided by the development of transgenic animal models in which expression of a specific T cell receptor (or B cell receptor) and its cognate self antigen is experimentally controlled. In most cases, expression of the self antigen was constitutive and did not allow for variation of its time‐ and dose‐dependent expression pattern, parameters which are known to influence the balance of tolerance versus immunity. We describe a transgenic model in which expression of human C‐reactive protein (hCRP), an acute‐phase protein, is tightly controlled at basal levels (female mice express around 10−9 M and male mice 5 × 10−7 M circulating hCRP) and is highly inducible (induction factor of 25–500). T cells from C57BL/6 mice recognize two epitopes of hCRP termed A (residues 79–95) and B (residues 87–102). Different efficacies of presentation in vitro and in vivo identify epitope A as subdominant and epitope B as dominant. T cells of non‐induced hCRP transgenic mice are tolerant to the dominant epitope, but reactive to the subdominant epitope. A hCRP‐specific IgG antibody response is detectable in transgenic mice, but is weaker than in littermates. Upon induction of hCRP, both T cell epitopes are presented by thymic and splenic antigen‐presenting cells (APC) in vivo. Kinetics of presentation by splenic APC closely match serum kinetics of hCRP, whereas presentation in the thymus is considerably prolonged. This model enables epitope‐specific T cell tolerance to be studied as a function of time‐ and dose‐dependent expression of the self antigen.
Identification of Sp1‐binding sites in the CD11c (p150,95α) and CD11a (LFA‐1α) integrin subunit promoters and their involvement in the tissuespecific expression of CD11c
Authors: López‐Rodríguez Cristina , Chen Hui‐Min , Tenen Daniel G. , Corbí Angel L.
Published: March 2007

The leukocyte integrins LFA‐1 (CD11a/CD18) and p150,95 (CD11c/CD18) mediate cell‐cell and cell‐extracellular matrix interactions during inflammatory responses and signal transduction into the cytoplasm. While the CD11a integrin subunit is expressed on all leukocytes, CD11c is almost exclusively expressed on cells of the myeloid lineage and on activated B lymphocytes. Its expression is regulated during cell activation and differentiation by transcriptional mechanisms. We have previously demonstrated that the proximal region of the CD11c promoter directs tissue‐restricted and developmentally‐regulated expression of reporter genes. Structural studies by electrophoretic mobility shift assays have demonstrated the presence of two Sp1‐binding sites at −70 (Sp1–70) and−120 (Sp1–120) which mediate the Sp1 transactivation of the CD11c promoter in Sp1‐defective SL2 cells, and which are involved in cell lineage‐specific DNA‐protein interactions, as demonstrated by footprinting in vivo. More importantly, mutation of either Sp1 site inhibited the activity of the CD11c promoter both in myeloid U937 cells and the CD11c‐expressing B lymphoblastoid JY cell line, while the opposite effect was observed in the CD11c‐negative epithelial HeLa cell line, demonstrating the involvement of both Sp1‐binding sites in the basal and the tissue‐restricted expression of the CD11c integrin subunit gene. Interestingly, the analysis of the CD11a proximal promoter also revealed the existence of an Sp1‐binding site at−70, indicating a common role for these cis‐acting elements in the transcription of the leukocyte integrin α subunit genes. The binding of Sp1 to the regulatory regions of the leukocyte integrin genes raises the possibility that the retinoblastoma susceptibility gene product is implicated in integrin expression through its functional interaction with Sp1, thus establishing a link between integrin‐dependent leukocyte adhesiveness and the state of cellular differentiation/proliferation.
CD4‐negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8‐deficient mice
Authors: Dalloul Ali H. , Ngo Karen , Fung‐Leung Wai‐Ping
Published: March 2007

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I‐disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC‐I or MHC‐II‐disparate, respectively). CD8–/– mice rejected MHC‐I‐disparate skin rapidly even if they were depleted of CD4+ cells in vivo (and were thus deficient in CD4+ and CD8+ T cells). By contrast, CD8+/+ controls depleted of CD4+ and CD8+ T cells in vivo accepted the MHC‐I‐disparate skin. Following MHC‐I, but not MHC‐II stimulation, allograft‐specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4−8−3intermediateTCRα/βintermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4+ cells in CD8–/– mice or of CD8+ cells in CD8+/+ mice. Our data suggest that this CD4−8−T cell population is responsible for the CTL function we have observed. Therefore, MHC class I‐restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up‐regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.
Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistance to a normally pathogenic challenge
Authors: Menon Juthika N. , Bretscher Peter A.
Published: March 2007

BALB/c mice are susceptible to a high‐dose infection of the protozoan Leishmania major, which induces a parasite‐specific antibody, Th2‐like response, exclusive of a significant and protective cell‐mediated Th1 component. We have shown, in contrast, that infection with a low number of parasites induces cell‐mediated immunity exclusive of antibody production, and results in resistance to substantial subsequent high‐dose infection. Low‐dose exposure thus constitutes effective vaccination. In the present study, we analyze lymphokine production by parasite‐specific T cells from these low‐dose exposed, resistant mice and from normal, susceptible mice following high‐dose infection. Two findings stand out. First, the parasite‐specific T cells in mice rendered resistant appear not to be in an activated, effector state at the time of parasite challenge, as assessed by lack of lymphokine production on short‐term stimulation with parasite antigens, but to be rather in a memory state. Second, the ratio of parasite antigen‐dependent production of interferon‐γ to that of interleukin‐4 by spleen cells of low‐dose exposed and normal mice upon high‐dose challenge takes a dramatically different course. This ratio is similar in both groups of mice shortly after challenge, but increases dramatically in the resistant and declines dramatically in the control mice over a period of weeks, such that these ratios differ by about 60‐fold 12 weeks after the high‐dose challenge. In addition, we show that a similar state of resistance occurs following low‐dose infection with a more virulent strain of L. major. In toto, our observations suggest that resistance may be generally achievable by low‐dose exposure and may be associated with a memory state which, when activated by parasite challenge, results in the evolution of the response over weeks such that the protective, Th1 component becomes ever more dominant over the Th2 component.
CD30 cell expression and abnormal soluble CD30 serum accumulation in Omenn's syndrome: Evidence for a T helper 2‐mediated condition
Authors: Chilosi Marco , Facchetti Fabio , Notarangelo Luigi D. , Romagnani Sergio , Del Prete Gianfranco , Almerigogna Fabio , De Carli Marco , Pizzolo Giovanni
Published: March 2007

Omenn's syndrome (OS) is a severe immunodeficiency, characterized by clinical and laboratory features reminiscent of a T helper type‐2 (Th2) response. CD30, a member of the tumor necrosis factor receptor superfamily, has been found to be preferentially expressed by human T cell clones exhibiting a Th2‐like profile and function. We investigated whether there are derangement in CD30 expression in tissues, and/or abnormalities in soluble CD30 (sCD30) levels in the serum, or both, of three children with OS and one child with maternal engraftment and Omenn's‐like syndrome (OLS). Large proportions of tissue‐infiltrating T lymphocytes from all four patients expressed CD30, whereas in control tissues, including peripheral blood, CD30+ T lymphocytes were extremely few or absent. In addition, levels of sCD30 were abnormally increased in all patients' sera. T cell clones were generated from sorted CD30+ and CD30− peripheral blood T cells of the patient with OLS who showed unusually high numbers of circulating CD30+ T lymphocytes. Most CD4+ T cell clones derived from CD30+ cells showed a Th2‐like cytokine profile, whereas the majority of clones generated from CD30− T cells were Th1. These findings support the hypothesis that Th2 cells are involved in the pathogenesis of OS. Moreover, they provide evidence that detection of CD30+ T cells in tissues, increased levels of sCD30 in biological fluids, or both, reflect the presence of immune responses characterized by prevalent activation of T cells producing Th2 cytokines.
Interferon‐γ and interleukin‐10 inhibit antigen presentation by Langerhans cells for T helper type 1 cells by suppressing their CD80 (B7‐1) expression
Authors: Ozawa Hiroaki , Aiba Setsuya , Nakagawa Satoshi , Tagami Hachiro
Published: March 2007

CD80(B7‐1) and CD86(B7‐2) co‐stimulatory molecules have been reported to activate Th1/Th2 development pathways differentially. It is well known that Langerhans cells (LC), potent antigen‐presenting dendritic cells in the epidermis, express several co‐stimulatory molecules and that this expression is modulated by several cytokines. Based on the recently reported effect of interferon (IFN)‐γ and interleukin (IL)‐10 on the expression of CD80 and CD86 by LC, we examined the effects of these cytokines on the expression of CD54 (intercellular adhesion molecule‐1) and CD40 in addition to CD80 and CD86 on LC, and correlated the expression of each co‐stimulatory molecule with antigen presentation for a Th1 clone by cultured LC (cLC) treated with these cytokines. LC cultured for 72 h significantly up‐regulated MHC class II antigen expression and all the co‐stimulatory molecules we examined. As previously reported, IL‐10 or IFN‐γ inhibited the up‐regulation of CD80 expression. Granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) partially restored the suppression of CD80 expression induced by IFN‐γ on cultured LC, while it had virtually no effect on the inhibition induced by IL‐10. Antigen presentation for the myoglobin‐specific syngeneic Th1 clone by cLC, which were pre‐incubated with these cytokines, correlated well with their CD80 expression. In addition, among the antibodies for CD80, CD86, CD28 or CD40, the suppression of the Th1 clone stimulation by LC was found to occur only with anti‐CD80 and anti‐CD28 antibodies. Finally, we studied the effects of IFN‐γ and IL‐10 on GM‐CSF production by epidermal keratinocytes (KC). We could show that only IFN‐γ, but not IL‐10, suppressed GM‐CSF production by KC. These findings suggest that both IFN‐γ and IL‐10 suppress antigen presentation by LC for Th1 cells by suppressing their CD80 expression. The inhibitory effect of IFN‐γ on CD80 expression on LC appears to be partially mediated through the suppression of GM‐CSF production by KC.
Permissive recognition during positive selection
Authors: Pawlowski Tomasz J. , Singleton Michael D. , Loh Dennis Y. , Berg Rance , Staerz Uwe D.
Published: March 2007

In the periphery αβ T lymphocytes recognize antigens in conjunction with major histocompatibility complex (MHC) molecules. In the thymus immature T cells are positively selected on MHC molecules in the apparent absence of cognate peptides. Thus, at different developmental stages a T cell responds to different epitopes, yet uses the identical αβ T cell antigen receptor (TcR). To explain this paradox it has been hypothesized that during positive selection immature T cells see peptides/ligands unique to the thymus, are selected by specific antagonists related to their cognate peptides, or are driven by lowered affinity thresholds of their TcR. Though different in detail, these theories rely on defined peptides uniquely matched to select certain TcR. However, we find that in a TcR‐transgenic (TcRtrans+) mouse severely limiting the diversity of peptides does not impair positive selection. We show that many unrelated peptides, including some naturally occurring on the cell surface, induce maturation of CD4−CD8+TcRhigh thymocytes. The same peptides when presented in conjunction with the selecting MHC molecule, are not recognized by peripheral T cells expressing the same TcRtrans. Therefore, these findings point to a promiscuous rather than discriminate recognition mode used by immature T cells.
Expression and function of B7‐1 and B7‐2 in hapten‐induced contact sensitivity
Authors: Reiser Hans , Schneeberger Eveline E.
Published: March 2007

We analyzed the expression and function of the co‐stimulatory molecules B7‐1 (CD80) and B7‐2 (CD86) during contact sensitivity reactions induced by the hapten 2,4‐dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7‐2. In contrast, following challenge with DNFB, expression of B7‐2 is up‐regulated in both epidermis and dermis. Importantly, B7‐1 is induced later and at lower levels compared to B7‐2. Intravenous injections of anti‐B7‐2 mAb, but not anti‐B7‐1 mAb partially inhibit the hapten‐induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti‐B7‐2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7‐2 is important for successful antigen priming.
Inhibition of tumor necrosis factor activity minimizes target organ damage in experimental autoimmune uveoretinitis despite quantitatively normal activated T cell traffic to the retina
Authors: Dick Andrew D. , McMenamin Paul G. , Körner Heinrich , Scallon Bernard J. , Ghrayeb John , Forrester John V. , Sedgwick Jonathon D.
Published: March 2007

Recent studies demonstrated that administration of a p55‐tumor necrosis factor (TNF) receptor IgG‐fusion protein (TNFR‐IgG) prevented the clinical onset of experimental autoimmune encephalomyelitis but did not alter the number or tissue distribution of autoantigen‐specific CD4+ effector T cells which trafficked into the central nervous system. To determine whether specific target tissues of autoimmune damage remain intact after TNFR‐IgG treatment despite the presence of inflammatory cells within the tissues, we examined rats with experimental autoimmune uveoretinitis (EAU), as in this model, the main target of autoreactive CD4+ T cells, the retinal rod outer segments (ROS), can be examined readily by light microscopy. As judged by direct ophthalmoscopy, the onset of inflammation in the anterior chamber of the eye in EAU following administration of TNFR‐IgG was delayed by 6 days compared to untreated controls, but the magnitude of the response was only slightly less than controls. Histological examination of the retinae and direct assessment of retinal inflammation revealed a disproportionate sparing of ROS in the TNFR‐IgG‐treated animals despite a level of retinal inflammation not substantially less than controls in which ROS damage was marked. Analysis of retinal leukocytes by immunofluo‐rescence microscopy and flow cytometry indicated that approximately equal numbers of CD4+ αβTCR+ lymphocytes were present in treated and control retinae, more than 30% of CD4+ cells in both experimental groups expressed the CD25 or MRC OX40 activation markers and most cells, which would include the CD4+ T lymphocytes, were activated as evidenced by MHC class II expression. Fewer activated macrophages and granulocytes were present in the treated retinae, possibly reflecting the lower level of tissue damage and subsequent accumulation of these inflammatory cells. The results demonstrate directly that a tissue specifically targeted for autoimmune destruction can be protected despite the influx of fully activated CD4+ T cells.
Fas‐mediated apoptosis is modulated by intracellular glutathione in human T cells
Authors: Chiba Takayuki , Takahashi Shuji , Sato Noriyuki , Ishii Seiichi , Kikuchi Kokichi
Published: March 2007

Fas antigen is a member of the tumor necrosis factor receptor family that transduces a lethal signal to the Fas‐sensitive cells. We previously established the Fas‐resistant variant cell lines LAC2D1R and JKT2D1R from the parental Fas‐sensitive cell lines, SUPT13 and Jurkat, respectively. Recently, we isolated the Fas‐resistant variant CEM2D1R from CCRF‐CEM. All of the variants were Fas+ but resistant to Fas‐mediated apoptosis. Further biochemical analysis revealed that the intracellular gluthathione (GSH) content of the Fas‐resistant variants was higher than in the original cells. When the Fas‐resistant variants were incubated with buthionine sulfoximine (BSO) or in GSH‐free/cysteine‐free medium to deplete GSH, Fas resistance was reversed. Incubation of the cells with cycloheximide also decreased intracellular GSH and reversed the Fas resistance. Furthermore, incubation of activated peripheral blood lymphocytes with BSO enhanced Fas‐mediated apoptosis. When the Fas‐sensitive cells were incubated with N‐acetylcysteine (NAC), intracellular GSH was increased and Fas‐mediated apoptosis was blocked. In contrast, Fas‐resistant variants, as well as Fas‐sensitive cells pre‐treated with NAC remained susceptible to allogeneic lymphokine‐activated killer cells, most likely due to perforin‐dependent killing. The results suggest that Fas‐mediated apoptosis, but not perforin‐dependent killing, is modulated by intracellular GSH in human T lymphocytes.
CD4‐dependent and ‐independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes
Authors: Criado Gabriel , Feito Maria José , Rojo José M.
Published: March 2007

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3‐TcR complex in stimulated or unstimulated cells, including p56lck, p59fyn and ZAP‐70. We have observed, in one mouse T helper CD4 T cell line, that most TcR‐ or CD3‐associated tyrosine kinase activity comes from CD4:p56lck (Diez‐Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4+ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4+ T lymphocytes. Our results show that, in normal CD4+ T lymphocytes, as in CD4+ T cell lines, there is a stable and readily detectable association between CD4: p56lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4+ lymphocytes have detectable amounts of p56lck associated in a CD4‐independent manner, as shown by immunodepletion of the lysates with anti‐CD4 antibodies. In addition, TcR/CD3 also bind p59fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co‐precipitates small quantities of p56fyn in a TcR/CD3‐independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4+ T cells and CD4+ T cell lines in kinase association to the TcR/CD3 complex.
Signal extinction and T cell repolarization in T helper cell‐antigen‐presenting cell conjugates
Authors: Valitutti Salvatore , Müller Sabina , Dessing Mark , Lanzavecchia Antonio
Published: March 2007

We have previously demonstrated that in T cell‐antigen‐presenting cell (APC) conjugates many T cell receptors (TCR) are serially triggered by a few peptide‐MHC complexes, resulting in sustained signaling. Here, we investigate the mechanisms that determine the duration and extent of signaling. We show that in the course of the T helper cell‐APC interaction, down‐regulation of triggered TCR leads to extinction of signaling. However, T cells that have been activated by a previous encounter with peptide‐pulsed APC and have extinguished signaling can swiftly repolarize towards APC displaying higher antigen concentrations and dedicate their help to these cells. These results demonstrate that TCR down‐regulation allows T cells to calibrate their response and dedicate their help to APC offering the highest stimulus.
Immunity to viruses in B cell‐deficient mice: Influence of antibodies on virus persistence and on T cell memory
Authors: Bründler Marie‐Anne , Aichele Peter , Bachmann Martin , Kitamura Daisuke , Rajewsky Klaus , Zinkernagel Rolf M.
Published: March 2007

Mice rendered B cell deficient by targeted disruption of the immunoglobulin μ chain gene (IgM−/− mice) were used to analyze the role of antibodies and B cells in viral infections; homozygous IgM−/− mice were bred in a way to avoid transmission of maternal antibodies. After infection with vesicular stomatitis virus (VSV), IgM−/− mice developed paralytic disease and subsequently died, whereas C57BL/6 control mice or IgM−/− mice passively protected with VSV‐neutralizing antibodies survived. Furthermore, IgM−/− mice showed increased natural killer (NK) activity upon exposure to either lymphocytic choriomeningitis virus (LCMV) or to poly(I) · poly(C), while NK activity in untreated IgM−/− mice was within normal ranges. Cytotoxic T cell responses were comparable in IgM−/− and control mice infected either with VSV or with vaccinia virus or with low doses of LCMV (102 infectious focus‐forming units [ifu]). After intracerebral infection with LCMV‐Armstrong, CD8+ T cell‐mediated lethal lymphocytic choriomeningitis developed independently of the presence of B cells and antibodies. After infection with high doses (2 × 106 − 5 × 106 ifu) of LCMV‐WE or LCMV‐Docile, IgM−/− mice exhibited a reduced capacity to control these primary infections and had elevated virus liters for prolonged times (>60 days). Nevertheless, the cytotoxic T cell response against LCMV in the early phase of infection was comparable in IgM−/− and control mice, but disappeared in those IgM−/− mice which had a persistent viral infection. Cytotoxic T cell memory was apparently unimpaired in low‐dose‐primed IgM−/−mice, which were able to control the primary virus infection; both IgM−/− and control mice cleared a high intravenous dose of virus within 2 days after challenge infection. This indicates that an efficient T cell memory against LCMV was established in the absence of B cells.
Transcriptional and translational control of TNF‐α gene expression in human monocytes by major histocompatibility complex class II ligands
Authors: Espel Enric , Garcia‐Sanz Jose A. , Aubert Vincent , Menoud Véronique , Sperisen Peter , Fernández Neus , Spertini François
Published: March 2007

While non‐stimulated primary human monocytes exhibit very low levels of tumor necrosis factor (TNF)‐α mRNA, direct binding of the staphylococcal exotoxin toxic shock syndrome toxin‐1 (TSST‐1) to major histocompatibility complex (MHC) class II molecules results in a fast (peak 1 h after stimulation), transient induction (sevenfold) of TNF‐α mRNA. This induction correlates with a fourfold increase in transcription rates of the TNF‐α gene, as detected by run‐on assays, and does not require de novo protein synthesis. Mapping of DNase‐I hypersensitive sites (DHS) discloses two constitutive DHS, one located far upstream (within the TNF‐β promoter) and the other centered at −39 ± 40 bp relative to the major TNF‐α transcription start site, suggesting that the TNF‐α gene was transcriptionally competent even prior to MHC class II engagement. Furthermore, stimulation of human monocytes with either TSST‐1 or lipopolysaccharide increases the translational efficiency of TNF‐α mRNA, as shown by a shift in the distribution of this mRNA species in polysome gradients and the translation rates of TNF‐α measured by immunoprecipitation from cells pulsed with [35S] methionine. The increase in translation efficiency of TNF‐α mRNA is independent of the half‐life of TNF‐α transcripts, which under the conditions used is unchanged. Taken together, our data indicate that TNF‐α expression is tightly regulated by MHC class II ligands, both at the transcriptional and translational levels.
Tyrosine kinase‐dependent activation of human NK cell functions upon triggering through CD44 receptor
Authors: Galandrini Ricciarda , Piccoli Mario , Frati Luigi , Santoni Angela
Published: March 2007

We recently showed evidence of CD44‐mediated enhancement of natural killer (NK) cell cytotoxic activity and induction of intracellular Ca2+ flux. In this study, we evaluated whether CD44 plays a stimulatory role in NK cell functions, such as cytokine production and activation antigen expression. Our results indicate that ligation of the CD44 receptor results in the induction of expression of the CD69 surface activation antigen as well as in the enhancement of phorbol ester‐induced TNF‐α secretion. We report also evidence for the coupling of CD44 receptor to a protein tyrosine kinase(s) pathway. CD44 engagement rapidly stimulates the tyrosine phosphorylation of several cellular substrates. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of CD44‐stimulated phosphorylation, indicating that it activates tyrosine kinase(s). Furthermore, the drug also prevents CD44‐mediated TNF‐α production and CD69 expression. These findings indicate that protein tyrosine phosphorylation is an early and critical event in CD44‐mediated activation of NK cell functions.
Epitope selection in major histocompatibility complex class I‐mediated pathway is affected by the intracellular localization of an antigen
Authors: Yamazaki Hiroko , Tanaka Masato , Nagoya Masako , Fujimaki Haruka , Sato Kazuki , Yago Tadayuki , Nagata Toshi , Minami Mutsuhiko
Published: March 2007

We analyzed the mode of antigen presentation of an endogenous antigen localized in the cytoplasm or in the mitochondria. Pseudomonas aeruginosa PAO leucine‐, isoleucine‐, valine‐binding protein (LIVAT‐BP) encoded by the braC gene was used as a model antigen. Using mouse BALB/3T3 cells, we established two LIVAT‐BP transfectants by transfection of a plasmid harboring the intact braC or braC gene fused with the mitochondrial transport signal derived from the yeast COXIV gene. One of the resulting transfectants, BC‐15, expressed LIVAT‐BP in the cytoplasm, while YZ‐710 cells expressed LIVAT‐BP in the mitochondria. The splenic effector cells derived from BALB/c mice primed with BC‐15 cells exhibited cytotoxic T lymphocyte (CTL) activity against BC‐15 cells, but not against YZ‐710 cells, whereas splenic effector cells primed with YZ‐710 cells exhibited CTL activity against YZ‐710 cells, but not against BC‐15 cells. Neither group of splenic effector cells showed CTL activity against parental BALB/3T3 cells. These CTL belonged to the CD8+ αβ T cell subset. Furthermore, we observed that the CTL activity against BC‐15 cells or YZ‐710 cells was blocked with anti‐H2‐Kd mAb, but not with anti‐H2‐Dd or H2‐Ld mAb. The CTL against BC‐15 or YZ‐710 cells could kill parental BALB/3T3 cells in the presence of peptides produced by alkali lysis of the LIVAT‐BP. suggesting that these CTL indeed recognized the peptide(s) derived from LIVAT‐BP. We determined that the epitope for the CTL against BC‐15 cells was QYGEGIATEV, corresponding to residues 162–171, and that the epitope recognized by the CTL against YZ‐710 cells was GYKLIFRTI, corresponding to residues 123–131 of LIVAT‐BP, respectively. Thus, we show here that epitope selection for MHC class I expression is affected by the intracellular localization of the antigenic protein.
Clonal analysis of immunoglobulin mRNA in rheumatoid arthritis synovium: Characterization of expanded IgG3 populations
Authors: Williams David Gareth , Taylor Peter Charles
Published: March 2007

Plasma cells secreting IgG, M, and A abound in the synovium of patients with rheumatoid arthritis, yet their immunoglobulin repertoire and clonal relationship remain to be elucidated. Locally synthesized immunoglobulins probably contribute to the chronic joint inflammatory processes which are characteristic of these patients. To determine whether B lymphocyte proliferation contributes to the synovial plasma cell infiltrate, the clonality of IgG mRNA in individual synovial biopsies from an actively inflamed joint of patients with rheumatoid arthritis was investigated by a combination of cDNA length analysis and DNA sequencing. Particular sizes of immunoglobulin cDNA, detectable in subclasses 1, 3, or 4, were expressed in most synovial biopsies from one patient, suggesting their origin from expanded clones present in each biopsy. To prove a clonal relationship between recurrent cDNA lengths, immunoglobulin cDNA was cloned from three regions of synovium in three patients. The sequence of clones with a recurrent cDNA length was determined. An IgG3 clone found in most synovial biopsies of one patient was encoded by an unmutated copy of the VH1 gene, DP7. In contrast, IgG3 clones encoded by mutated versions of the VH3 gene DP49 or the VH4 gene DP63 were expanded in the other two patients. Different somatic mutants of these clones were isolated from different sites in these patients. The ratio of replacement/silent somatic mutations in these two families of clones suggests that the selective clonal expansion in the synovium of patients with rheumatoid arthritis is due to an antigen‐driven immune response.