bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma
Authors: Bogen Bjarne
Published: March 2007

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)‐transgenic mice were injected with plasmacytoma cells secreting a highly tumor‐specific antigen, a monoclonal immunglobulin (Ig), for which the transgene‐encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen‐presenting cells. The TCR‐transgenic mice have previously been shown to be protected against an Id+ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id‐specific CD4+ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4+ cells express endogenous, rather than transgene‐encoded TCR α chains. Peripheral deletion, functional T cell unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, ∼50 μg/ml (0.3 μM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.
Conservation of minor histocompatibility antigens between human and non‐human primates
Authors: den Haan Joke M. M. , Bontrop Ronald E. , Pool Jos , Sherman Nicholas , Blokland Els , Engelhard Victor H. , Hunt Donald F. , Goulmy Els
Published: March 2007

It is well accepted that minor histocompatibility antigens (mHag) can function as transplantation barriers between HLA‐matched individuals. Little is known about the molecular nature and evolutionary conservation of mHag. It is only very recently that the first human mHag were identified. The HLA‐A2.1‐restricted mHag HA‐2 and the HLA‐B7‐restricted mHag H‐Y appeared to be peptides derived from polymorphic self proteins. Here we show that the HLA‐A2.1‐restricted mHag HA‐1, HA‐2, and the H‐Y peptides are conserved between man, chimpanzees and rhesus macaques. Human cytotoxic T cell clones specific for the HLA‐A2.1‐restricted mHag HA‐1, HA‐2, and H‐Y recognized HLA‐A2.1 gene‐transfected chimpanzee and rhesus macaque cells. Highpressure liquid chromatography fractionation of HLA‐A2.1‐bound peptides isolated from the HLA‐A2.1‐transfected chimpanzee cells revealed that the chimpanzee HA‐1 and HA‐2 co‐eluted with the human HA‐1 and HA‐2. Subsequent amino acid sequencing showed that the chimpanzee HA‐2 peptide is identical to the human HA‐2 peptide. Our functional and biochemical results demonstrate that mHag peptides are conserved for over 35 million years.
Induction of CD14 expression in Lpsn, Lpsd and tumor necrosis factor receptor‐deficient mice
Authors: Takakuwa Tetsuya , Knopf Hans‐Peter , Sing Andreas , Carsetti Rita , Galanos Chris , Freudenberg Marina A.
Published: March 2007

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lpsd mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsn (LPS sensitive) and Lpsd mice, and its modulation by LPS, killed gramnegative and gram‐positive bacteria and double‐stranded (ds)RNA. Untreated Lpsn and Lpsd cultured macrophages (MΦ), expressed similar amounts of CD14 mRNA and membrane‐bound (m)CD14. LPS enhanced CD14 expression only in Lpsn MΦ, while all bacteria, or dsRNA, enhanced CD14 in Lpsn and Lpsd MΦ. Similarly, in vivo administration of LPS induced or enhanced CD 14 mRNA in different organs of Lpsn mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD 14 expression in Lpsn, Lpsd and also in TNF receptor 2‐deficient (TNFR2−/−) mice, but failed to do so in TNFR1−/− mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2−/− and TNFR1−/− mice to a similar extent, revealing that this induction does not require TNF signaling.
Immunodominance of cytotoxic T lymphocyte epitopes co‐injected in vivo and modulation by interleukin‐12
Authors: Eberl Gérard , Kessler Benedikt , Eberl Lucie Peduto , Brunda Michael J. , Valmori Danila , Corradin Giampietro
Published: March 2007

Immunodominance (ID) of T cell epitopes is a well‐documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H‐2Kd, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half‐life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co‐injected CTL epitopes can be determined both at the peptide level (binding affinity to H‐2Kd) and at the T cell level. This hypothesis is supported by our finding that interleukin‐12 strongly modulates ID when it is not correlated with MHC binding.
Differential regulation of interleukin‐12‐and interleukin‐15‐induced natural killer cell activation by interleukin‐4
Authors: Salvucci Ombretta , Mami‐Chouaib Fathia , Moreau Jean Louis , Thèze Jacques , Chehimi Jihed , Chouaib Salem
Published: March 2007

The regulation of human natural killer (NK) cell activation is under the control of a network of regulatory signals provided by cytokines. In the present study, we investigated the functional interaction between interleukin (IL)‐4 and two monocyte/macrophage‐derived cytokines, IL‐12 and IL‐15, during the process of NK stimulation. Using freshly isolated human NK cells, we have demonstrated that IL‐4 negatively regulates lymphokine‐activated killer (LAK) activity induced by IL‐15 against the NK‐resistant Daudi target cells. In contrast, IL‐4 had no effect on IL‐12‐stimulated LAK generation. The differential effect of IL‐4 on NK cell activation by IL‐12 and IL‐15 correlates with its ability to increase or to down‐regulate the level of tumor necrosis factor‐α and interferon‐γ release by NK cells, respectively. In contrast, endogenous transforming growth factor‐β1 does not appear to be involved in the IL‐4 regulatory pathway. Furthermore, while IL‐4 was found to decrease the basal expression of the IL‐2 receptor β subunit utilized by IL‐15, it had no effect on the expression of the β1 chain of the IL‐12 receptor compared to untreated cells. Northern blot analysis indicated that the IL‐4 regulatory effect on NK lytic function was associated with its capacity to down‐regulate granzyme B and perforin gene transcription in response to IL‐15 and its failure to affect the expression of both gene's in response to IL‐12. Together, these data suggest the existence of a distinct cross‐talk between IL‐4 and IL‐15 or IL‐12 signaling pathways during the regulation of human non‐major histocompatibility complex‐restricted cytotoxicity.
Autoimmune disease of exocrine organs in immunodeficient alymphoplasia mice: a spontaneous model for Sjören's syndrome
Authors: Tsubata Rieko , Tsubata Takeshi , Hiai Hiroshi , Shinkura Reiko , Matsumura Ryutaro , Sumida Takayuki , Miyawaki Shigeki , Ishida Hiroshi , Kumagai Shunichi , Nakao Kazuwa , Honjo Tasuku
Published: March 2007

Mice homozygous for an autosomal recessive mutation aly (alymphoplasia) lack both lymph nodes and Peyer's patches, and show defects in both humoral and cellular immunity. Histopathological analysis revealed chronic inflammatory changes in exocrine organs such as the salivary gland, lacrimal gland, and pancreas of the homozygotes (aly/aly), but not the heterozygotes (aly/+). In these exocrine organs, mononuclear cells consisting mainly of CD4+ T cells infiltrate periductal areas, and, in some cases, the cell infiltration extended to lobules. The inflammatory changes in exocrine organs were transferred by a T cell‐enriched fraction of spleen cells from homozygous animals. These results suggest that autoimmune mechanisms mediated by self‐reactive T cells may be involved in the inflammatory lesions of various exocrine organs in the homozygous mice, although these mice show immunodeficiency. Inflammatory changes were also observed in the lung of the homozygotes. Since Sjögren's syndrome is characterized by diffuse lymphocyte infiltration in the periductal areas of the lacrimal and salivary glands and is occasionally associated with pulmonary disease, aly/aly mice may serve as a unique spontaneous model of Sjögren's syndrome.
Thymocyte differentiation in γ‐irradiated severe‐combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor α locus
Authors: Zhu Chengming , Bogue Molly A. , Roth David B.
Published: March 2007

Treatment with DNA‐damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe‐combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) β, γ and δ loci, but not at the TCR α locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation‐mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR α locus. We found that Jα‐coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4+CD8+ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR α is sufficient for irradiation‐mediated rescue of coding joint formation, we administered a second dose of γ‐irradiation after abundant CD4+ CD8+ thymocytes and hairpin TCR α coding ends had accumulated. This treatment failed to stimulate rescue of TCR α coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation‐mediated rescue of TCR rearrangements in SCID mice.
Major histocompatibility complex and T cell receptor interaction of the P91A tum− peptide
Authors: McCormick David , Stauss Hans J. , Thorpe Christopher , Travers Paul , Dyson P. Julian
Published: March 2007

The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA 700 proteasome regulatory complex. P91A stimulates a strong CD8+ T cell response when expressed on tumor cells or normal tissue and P91A‐specific T cells express a restricted range of T cell receptors. Although it is a strong Ld‐binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. Ld predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the Ld motif residue proline at position 2, residues 5–7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with Ld. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum+ peptide (QNRRALDL) to bind to Ld is addressed by molecular modeling.
Transgene‐encoded human CD2 acts in a dominant negative fashion to modify thymocyte selection signals in mice
Authors: Melton Elizabeth , Sarner Nitza , Torkar Michaela , van der Merwe P. Anton , Russell Jennifer Q. , Budd Ralph C. , Mamalaki Clio , Tolaini Mauro , Kioussis Dimitris , Zamoyska Rose
Published: March 2007

CD2 is a cell surface glycoprotein present on all T cells which has been shown to function as an adhesion and signaling molecule. Expressed early in T cell development, human CD2 (HCD2) has been suggested to play a role during thymopoiesis. However, the relevance of CD2 in T cell development has been called into question recently, as neither disruption of the CD2 gene nor anti‐CD2 antibody treatment of fetal thymic organ cultures in mouse were shown to have any discernible consequences. We have expressed HCD2 at high levels in transgenic mice and found a profound effect of the transgene on thymocyte differentiation. Transgenic thymuses are considerably reduced in cell number as a consequence of increased apoptosis of double‐positive (DP) thymocytes in the cortex. The remaining DP cells have up‐regulated levels of T cell receptor (TCR) and are resistant to apoptosis mediated by administration of antigen. These effects are dependent on the cytoplasmic domain of HCD2, as mice expressing comparable levels of a tailless HCD2 transgene have a normal phenotype. The HCD2 cytoplasmic domain contains several regions of identity with mouse CD2 and can interact effciently with mouse intracellular signaling machinery. These results suggest there is considerable cross‐talk between CD2 and TCR on developing thymocytes with consequences for the stimulation threshold of mature T cells.
Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells
Authors: Samuelsson Astrid , Yari Fayezeh , Hinkula Jorma , Ersoy Oguz , Norrby Erling , Persson Mats A. A.
Published: March 2007

Human antibodies against HIV‐1 have been sought to study neutralization events on the molecular level, and for possible use in passive immune intervention. The development of phage display techniques has opened the possibility of rapidly generating human monoclonal antibodies with desired specificities. We and others have isolated human HIV‐1 neutralizing antibody fragments using this technique. Bacterial expression of isolated clones does, however, differ broadly both in expression levels and functional activity. In addition, intact IgG cannot be expressed in bacteria. By transferring the genes of isolated Fab clones to a mammalian expression system we could perform a comparison of functional activity between Fab expressed in bacterial and mammalian cells, as well as Fab and whole IgG. Fab fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in Escherichia coli, underlining the inefficiency of procaryotic expression. No difference in HIV‐1 neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells. Thus, bivalency does not always confer improved neutralization efficacy.
The development of IgE+ memory B cells following primary IgE immune responses
Authors: Le Gros Graham , Schultze Nathalie , Walti Simone , Einsle Karin , Finkelman Fred , Kosco‐Vilbois Marie H. , Heusser Christoph
Published: March 2007

We studied whether long‐lived IgE+ memory B cells develop following three types of primary IgE immune responses. Immunization of mice with anti‐IgD antibody induced a T cell‐dependent, interleukin (IL)‐4‐dependent primary IgE response and the formation of IgE isotype switched (IgE+) memory B cells. These IgE+ memory B cells could be stimulated in vivo by injection with goat anti‐IgE antibodies to produce a profound IL‐4‐independent memory IgE response. By contrast, both infection of mice with Nippostrongylus brasiliensis or repeated immunization with benzylpenicilloyl‐keyhole limpet hemocyanin (BPO‐KLH) in alum stimulated good primary IgE responses and profound memory T cell‐dependent antigen‐specific IgE responses, but failed to induce the development of long lived IgE+ memory B cells because they could not be recalled with goat anti‐IgE antibodies. Mice receiving double immunizations combining anti‐IgD with either N. brasiliensis infection or BPO‐KLH immunization mounted significant goat anti‐IgE‐induced secondary IgE responses, but no N. brasiliensis or BPO‐KLH‐specific IgE could be detected. This indicates that the N. brasiliensis and BPO‐KLH induced immune responses do not suppress the development of IgE+ B cells, but rather, do not provide the necessary conditions for their formation. Taken together these data indicate that long‐lived IgE+ B cells fail to develop during the primary IgE response to N. brasiliensis infection or BPO‐KLH immunization. By contrast, significant numbers of IgE+ memory B cells form during the primary IgE immune response induced by anti‐IgD immunization. Our observations suggest that immunization protocols involving membrane IgD cross‐linking and limited duration of cognate T cell help are necessary for the formation of IgE+ memory B cells. It will be important to determine the relevance of membrane IgD interaction with allergens, as this would influence the design of new therapies for the treatment of allergy and asthma.
CD40 engagement induces monocyte procoagulant activity through an interleukin‐10 resistant pathway
Authors: Pradier Olivier , Willems Fabienne , Abramowicz Daniel , Schandené Liliane , De Boer Mark , Thielemans Kris , Capel Paul , Goldman Michel
Published: March 2007

Interactions between membrane‐bound molecules were previously shown to be involved in the induction of tissue factor‐dependent monocyte procoagulant activity (PCA) by activated T cells. To investigate the potential role of the CD40/CD40 ligand (CD40L) pathway in this process, we first determined the effects of blocking anti‐CD40 or anti‐CD40L monoclonal antibodies (mAb) on the development of monocyte PCA during mixed lymphocyte reaction (MLR) between allogeneic peripheral blood mononuclear cells (PBMC). The strong inhibitory effect exerted by both mAb (mean percentages of inhibition: 88 and 91% for anti‐CD40 and anti‐CD40L mAb, respectively) indicates that CD40/CD40L interactions are required for the induction of PCA in MLR. These data led us to measure monocyte PCA after incubation of PBMC or purified monocytes with a stimulating anti‐CD40 mAb (BL‐C4) or with 3T6 fibroblasts transfected with the gene encoding CD40L. In both systems, we found that CD40 engagement strongly induced monocyte PCA which was related to tissue factor expression as shown by flow cytometric analysis. Finally, we observed that recombinant interleukin (IL)‐10, which inhibits lipopolysaccharide‐induced PCA, did not significantly influence CD40‐dependent PCA. We conclude that CD40 engagement on monocytes induces tissue factor‐dependent PCA through an IL‐10‐resistant pathway. These findings have implications for the control of coagulation events triggered by interactions between T cells and monocytes.
The canonical T cell receptor of dendritic epidermal γδ T cells is highly conserved between rats and mice
Authors: Kühnlein Petra , Mitnacht Rita , Torres‐Nagel Nora E. , Herrmann Thomas , Elbe Adelheid , Hünig Thomas
Published: March 2007

Two monoclonal antibodies with specificity for rat γδ T cell receptor (TCR) were generated. One, called V65, reacts with all CD3+ αβ TCR− rat T cells and thus recognizes a constant determinant of the rat γδ TCR (Kühnlein et al., Journal of Immunology 1994, 153: 979). The other, called V45, reacts with approximately 80% of γδ T cells in peripheral lymphoid organs. In rat epidermis, V65 but not V45 detects a dense network of the dendritic epidermal T cells (DETC). Analysis of epidermal RNA by polymerase chain reaction (PCR) indicated that Vγ3 and Vδ1 are the predominant, if not exclusive TCR V transcripts present at this site. Sequence analysis of cDNA clones obtained by reverse transcription‐PCR with Vγ3‐ and Vδ1‐specific primers revealed that the variable domains of rat DETC γ and δ chains are very homologous to those described in mice (92 % and 95 % identity at the protein level). The complete conservation between the two species of the amino acid sequences at the V‐(D)‐J transitions of this monomorphic receptor indicates that the interaction of the DETC TCR with its as yet unknown ligand must be of central importance for DETC function.
Inducible nuclear factors binding the IgM heavy chain pre‐mRNA secretory poly(A) site
Authors: Phillips Cathy , Schimpl Anneliese , Dietrich‐Goetz Walter , Clements J. Barklie , Virtanen Anders
Published: March 2007

Two alternative forms of IgM heavy‐chain mRNA are produced from a common precursor mRNA as a result of competition between cleavage/poly(A) addition at the upstream (secretory) poly(A) site and cleavage/poly(A) addition at the downstream (membrane) poly(A) site coupled with splicing. The efficiency of cleavage at the secretory poly(A) site is thought to play a crucial role in this alternative processing. We therefore examined RNA binding factors recognizing the secretory poly(A) site, in the absence of the splicing option, to look for transacting factors that may play a role in cleavage/polyadenylation efficiency at this site. Purified primary B cells produce the secretory form of μ mRNA when stimulated with lipopolysaccharide (LPS) and the membrane form of μ mRNA when their antigen receptors are ligated by anti‐μ antibodies. We compared RNA binding factors in nuclear extracts from cells produced by these different stimulatory conditions and show that induction of the secretory form of μ mRNA by LPS correlates with the induction of a 28–32‐kDa secretory poly(A) site‐specific polypeptide which is also present in the plasmacytoma cell line J558L. Visualization of the 28–32‐kDa polypeptide in UV cross‐linking assays depends on a GU‐rich element downstream of the secretory poly (A) site. We show that this GU‐rich region enhances polyadenylation efficiency in vivo by transfection of luciferase reporter constructs into the plasmacytoma J558L. We also examined nuclear extracts from B cells doubly stimulated with LPS and anti‐μ antibodies in which expression of the secretory form of μ mRNA is selectively inhibited. This inhibition may be due to a down‐regulation of polyadenylation at the secretory poly(A) site or an up‐regulation of the competitive splicing process. This form of stimulation does not lead to the disappearance of the 28–32‐kDa polypeptide, but to an enhanced binding of a 50–55‐kDa factor which binds both the secretory and membrane poly(A) site. We report the first detection of changes in RNA binding factors taking place at the secretory poly(A) site which correlate with the expression of different forms of μ mRNA produced by primary B cells under different stimulation conditions.
Antigen presentation by Leishmania mexicana‐infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface
Authors: Wolfram Markus , Fuchs Manuela , Wiese Martin , Stierhof York‐Dieter , Overath Peter
Published: March 2007

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow‐derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane‐bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface‐exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild‐type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild‐type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.
Leishmania‐infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells
Authors: Kima Peter E. , Soong Lynn , Chicharro Carmen , Ruddle Nancy H. , McMahon‐Pratt Diane
Published: March 2007

CD4+ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the 14.07 macrophage cell line (H‐2k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4+ T cells. In contrast, promastigote‐infected macrophages did present endogenous parasite molecules to CD4+ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post‐infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4+ T cells, a major and critical anti‐leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.
B lymphocytes are impaired in mice lacking the transcriptional co‐activator Bob1/OCA‐B/OBF1
Authors: Nielsen Peter J. , Georgiev Oleg , Lorenz Barbara , Schaffner Walter
Published: March 2007

The protein Bob1 (also called OCA‐B or OBF‐1) is a B lymphocyte‐specific transcriptional co‐activator which can stimulate transcription by interaction with Oct‐1 or Oct‐2 and the octamer sequence found in the promoter and most enhancer regions of the immunoglobulin genes. The role that Bobl plays in the maturation and function of B cells was investigated in mice deficient for Bobl1. Although early stages of B cell development in the bone marrow do not appear to be affected, these mice show reduced numbers of B cells in peripheral lymph‐oid organs. Based on staining for B220, heat‐stable antigen, and IgD, this reduction is particularly strong in those cells representing the more mature B cell stages. Probably as a consequence of this reduction in mature B cells, Bobl1‐deficient mice show reduced serum titers of the immunoglobulin isotypes IgG1, IgG2a, IgG2b and IgA, but not IgM. Histological examination of sections from spleen and lymph nodes reveal that while Bob‐1‐deficient mice have primary follicles, they lack well‐developed germinal centers. Interestingly, B1 (Ly1) B cells in the peritoneum do not appear to be affected by the lack of Bobl1. Taken together, these results suggest that, at least in conventional B cells, Bob1 plays in important role in the antigen‐driven stages of B cell activation and maturation.
Major histocompatibility complex class I‐dependent cell binding to isolated Ly‐49A: evidence for high‐avidity interaction
Authors: Chang Chew Shun , Shen Lianjun , Gong Dong‐Er , Kane Kevin P.
Published: March 2007

Ly‐49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H‐2Dd‐expressing target cells. In the present report, we immunoaffinity‐purified Ly‐49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly‐49A. Adhesion was observed by cells expressing relatively high levels of H‐2Dd, but not cells expressing very low or no cell surface Dd, while antibodies specific for Dd or Ly‐49A inhibited the cell binding, indicating that Dd and Ly‐49A mediate the observed adhesion. The density of immobilized Ly‐49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly‐49A density requirement, and above this threshold, increases in Ly‐49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly‐49A homodimers required to mediate cell adhesion was found to be quite low: 140–250 molecules/μm2. These results suggest that the avidity of Ly‐49A for Dd is relatively high and indicate that small changes in Ly‐49A density near the threshold result in large changes in stable Ly‐49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly‐49A receptor engagement may explain the observation that relatively small differences in Ly‐49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of Dd is spared from lysis or not.
Migratory response of human natural killer cells to lymphotactin
Authors: Giancarlo Bianchi , Silvano Sozzani , Albert Zlotnik , Alberto Mantovani , Paola Allavena
Published: March 2007

Lymphotactin (Lptn) is a new protein belonging to the C or γ subfamily of chemokines with only two of the four cysteine residues. Lptn was reported to act specifically on T lymphocytes and not on monocytes and neutrophils. To understand better the spectrum of action of Lptn we have examined its ability to induce natural killer (NK) cell migration. Freshly isolated human NK cells as well as long‐term cultured NK cells propagated in interleukin‐2 (IL‐2)‐containing medium migrated in response to Lptn. Optimal activity was observed at concentrations ranging from 50 to 200 ng/ml, and the efficacy was comparable to that of MCP‐1, the prototype of C‐C chemokines. Migration in response to Lptn was chemotaxis rather than chemokinesis as determined in a checkerboard analysis. Migration of NK cells was comparable to that observed with T lymphocytes from the same donor, under the same experimental conditions. Finally, in contrast to other cytokines (IL‐2 and IL‐12) which in addition to chemotaxis augment NK cell adhesion to endothelial cells in vitro, Lptn did not affect the adhesiveness of NK cells to vascular endothelium.
Human T lymphocytes bind to germinal centers of human tonsils via integrin α4/VCAM‐1 and LFA‐1/ICAM−1 and −2
Authors: Schriever Folke , Korinth Dirk , Salahi André , Lefterova Petja , Schmidt‐Wolf Ingo G. H. , Behr Susanne I.
Published: March 2007

Binding of T lymphocytes within the different compartments of the secondary lymphoid organs is crucial for the function of the cellular and the humoral immune response. It is still not known which adhesion molecules guide T cells to the distinct areas of the lymphoid microenvironment. In the current study an in situ adhesion assay was used to define the receptors for binding of T cells to human tonsils. The T cell lines Jurkat and MOLT‐4 and normal, activated T cells were found to bind exclusively to germinal centers. Jurkat cells used the receptor pair integrin‐α4 (VLA‐4α)/VCAM‐1, whereas activated MOLT‐4 cells and normal T cells bound via both adhesion pathways, namely via integrin‐α4/VCAM‐1 and LFA‐1/ICAM‐1 and ‐2. It is suggested that these adhesion mechanisms are involved in the migration of T cells into the germinal centers of secondary lymphoid organs and that they influence the selection of B cells by apoptosis.
Integrin α chain cytoplasmic tails regulate “antibody‐redirected” cell adhesion, independently of ligand binding
Authors: Weitzman Jonathan B. , Pujades Cristina , Hemler Martin E.
Published: March 2007

Here we describe a novel “antibody‐redirected cell adhesion” (ARCA) assay. This assay measures heterotypic cell‐cell adhesion, resulting from antibody bridging between Fcγ receptors type II (CD32) on leukocytes, and clustered intergrins on adherent cell monolayers. This ARCA activity, facilitated by integrins αβ1 or α4β1, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the α4 cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although α4 tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM‐1 to intact α4‐transfected K562 cells. In conclusion, the integrin α chain tail can positively regulate integrin‐dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand‐binding considerations.
Regulated production of the interferon‐γ‐inducible protein−10 (IP‐10) chemokine by human neutrophils
Authors: Cassatella Marco A. , Gasperini Sara , Calzetti Federica , Bertagnin Anna , Luster Andrew D. , McDonald Patrick P.
Published: March 2007

Interferon‐γ (IFN‐γ)‐inducible protein‐10 (IP‐10), a member of the C‐X‐C sub‐family of chemokines, is known to be produced by monocytes, lymphocytes, keratinocytes and endothelial cells in response to IFN‐γ Here, we show that human polymorphonuclear neutrophils (PMN) also have the ability to produce IP‐10. IFN‐γ alone had a modest effect on IP‐10 mRNA accumulation, whereas tumor necrosis factor‐α (TNF‐α), yeast particles opsonized with IgG (Y‐IgG), lipopolysaccharide (LPS), and formyl‐methionyl‐leucyl‐phenylalanine (fMLP) all failed to up‐regulate IP‐10 gene expression. However, stimulation of neutrophils with IFN‐γ in combination with either TNF‐α or LPS (but not with Y‐IgG or fMLP) resulted in a considerable induction of IP‐10 mRNA transcripts, as well as in the extracellular release of the protein. In contrast, the best inducer of IP‐10 release from peripheral blood mononuclear cells was IFN‐γ alone. Furthermore, mRNA stabilization analyses demonstrated that IP‐10 mRNA isolated from PMN stimulated with IFN‐γ only, or with IFN‐γ plus either TNF‐α or LPS, had similar half‐lives. Finally, we found that interleukin‐10, a known inhibitor of chemokine production in PMN, moderatley suppressed the extracellular production of IP‐10 in neutrophils stimulated with IFN‐γ plus either LPS or TNF‐α. Since IP‐10 is a potent chemoattractant for activated T lymphocytes, the ability of neutrophils to produce IP‐10 might contribute to the evolution and progression of the inflammatory response.
Pattern of usage and somatic hypermutation in the VH5 gene segments of a patient with asthma: Implications for IgE
Authors: Snow Rachel E. , Chapman Caroline J. , Frew Anthony J. , Holgate Stephen T. , Stevenson Freda K.
Published: March 2007

The VH5 family contains two functional genes, V5–51 and VH32, and appears to be over‐represented in IgE antibodies from patients with allergic disease. Previous sequence analysis of VH5 gene segments in IgE has revealed a substantial level of somatic hypermutation, with evidence for hotspots. To assess characteristics of VH5 gene behavior, VH5 gene segments in combination with Cμ, Cγ, Cα, and Cε have been amplified from blood B lymphocytes of a patient with atopic asthma. Sequence analysis revealed strong preferential usage of one of the two VH5 gene segments (V5–51) by IgM, IgG, and IgA. In contrast, IgE used both genes equally. Levels of somatic mutation were higher following all isotype switches, particularly to IgA. Mutational hotspots were identifiable in all isotypes, leading to several common replacement amino acids. The dominant mutational site in IgM was a common hotspot at Ser31. IgG, IgA, and IgE‐derived sequences had mainly common hotspots, with few distinct sites. The results indicate that mutational hotspots are a feature of the VH5 gene, are identifiable at an early stage of somatic hypermutation, and are not a unique feature of IgE. Generation of IgE antibodies appears to involve three processes: the preferential use of VH5 genes, consistent with superantigen stimulation; the accumulation of somatic mutations in common hotspots, some of which are in complementarity‐determining regions (CDR); and the acquisition of non‐hotspot mutations in CDR, accounting for ∼ 50% of replacement amino acids in these sites, and presumably contributing to affinity maturation.
Major histocompatibility complex recognition by immune receptors: Differences among T cell receptor versus antibody interactions with the VSV8/H‐2Kb complex
Authors: Witte Torsten , Smolyar Alex , Spoerl Rebecca , Goyarts Earl C. , Nathenson Stanley G. , Reinherz Ellis L. , Chang Hsiu‐Ching
Published: March 2007

The surface residues of the VSV8/Kb complex important for recognition by N15 and N26 αβ T cell receptors (TCR) were mapped by mutational analysis and compared to each other and with epitopes of well‐characterized Kb specific monoclonal antibodies (mAb). Three features of immune receptor recognition emerge. First, the footprints of the two TCR on VSV8/Kb are similar with more than 80% overlap between sites. Given that only 8 of 14 surface exposed VSV8/Kb residues identified as critical for TCR interaction are in common, the chemical basis of the N15 and N26 interactions is nevertheless distinct. Second, the cognate peptide is a major focus of TCR recognition: mutation at any of the three exposed side chains (at p1, p4 or p6) abrogates interaction of both TCR as measured by functional T cell activation. Third, in contrast to TCR, mAb bind to discrete segments on the periphery of the α1 and/or α2 helices without orientational restriction. These findings suggest that unlike soluble antibodies, surface membrane receptor‐ligand interactions on opposing cells (i.e. TCR‐peptide/MHC, CD8‐MHC) limit the orientational freedom of the TCR in the immune recognition process.
CD28‐mediated induction of proliferation in resting T cells in vitro and in vivo without engagement of the T cell receptor: Evidence for functionally distinct forms of CD28
Authors: Tacke Michael , Hanke Gabriele , Hanke Thomas , Hünig Thomas
Published: March 2007

JJ316 and JJ319 are rat CD28‐specific monoclonal antibodies (mAb) of the γ1χ isotype with identical co‐stimulatory potency. At a concentration 100–1000‐fold higher than that required for co‐stimulation, JJ316, but not JJ319 induces massive proliferation of all T cell subsets in vitro without T cell receptor (TCR) triggering. “Direct” stimulation by JJ316 is fully blocked by JJ319, indicating that it is not due to cross‐reactivity of JJ316 with the TCR complex or other activating receptors. JJ316 binds much more slowly to primary T cells than JJ319, whereas both antibodies bind with similar kinetics to CD28‐transfected L‐929 cells, suggesting that JJ316 binding to T cells requires redistribution or a conformational change of CD28. In vivo, JJ316 but not JJ319 induces rapid and transient proliferation of most CD4 T cells and, indirectly, of B cells. These data show that TCR engagement is not an absolute prerequisite either in vitro or in vivo for the induction of T cell proliferation through CD28 and suggest that mAb JJ316 is able to stimulate resting T cells directly by recruiting CD28 molecules from an inactive to an active form.
Accumulation of somatic hypermutation and antigen‐driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary antihapten response in mice
Authors: Kimoto Hiroshi , Nagaoka Hitoshi , Adachi Yoshihiro , Mizuochi Toshiaki , Azuma Takachika , Yagi Takenori , Sata Tetsutaro , Yonehara Shin , Tsunetsugu‐Yokota Yasuko , Taniguchi Masaru , Takemori Toshitada
Published: March 2007

Well‐developed germinal centers (GC) contain rapidly dividing surface immunoglobulin‐negative (sIg‐) B cells (centroblasts), and most of their progeny are sIg+ B cells (centrocytes) in a resting state. It has been predicted that somatic hypermutation occurs in centroblasts, whereas antigen‐driven selection takes place in centrocytes. The present analysis indicates that murine GC B cells bearing sIg with specificity for an immunizing antigen are in a rapidly cycling state and increase exponentially in number to occupy spleen GC at high frequency during the 1st week after primary immunization; however, the number of these cells is significantly reduced in the 2nd week of immunization. During that period, these proliferating sIg+ GC B cells accumulate somatic hypermutations with nucleotide exchanges indicative of affinity maturation. These sIg+ GC B cells co‐express B7‐2, ICAM‐1, and LFA‐1, and have potent antigen‐presenting activity which results in T cell activation in vitro. These observations indicate that the sIg+ GC B cells accumulate somatic hypermutations and undergo antigen‐driven selection through proliferation, probably upon activation by T cells. This sIg+ GC B cell population may represent cell cycling centrocytes; however, the possibility that these may represent centroblasts undergoing re‐expression of sIg could not be excluded.
Redox regulation of apoptosis: Impact of thiol oxidation status on mitochondrial function
Authors: Marchetti Philippe , Decaudin Didier , Macho Antonio , Zamzami Naoufal , Hirsch Tamara , Susin Santos A. , Kroemer Guido
Published: March 2007

The probability that a cell will undergo apoptosis is in part dictated by the cellular redox potential, which is mainly determined by the reduction and oxidation of thiol residues on glutathione and proteins. We and others have recently shown that mitochondria play a critical role in the apoptotic cascade. Here, we address the question as to whether thiol modification regulates apoptosis and in which cellular compartment apoptosis‐regulatory thiols are localized. To resolve this problem, we employed the divalent thiol‐reactive agent diamide, which causes thiol cross‐linking and thus mimics disulfide bridge formation, and a panel of monovalent thiol‐reactive compounds (which impede disulfide bridge formation due to thiol oxidation), one of which is specifically targeted to the mitochondrial matrix. Our data indicate that thymocyte apoptosis induced by diamide mimics natural apoptosis in the sense that mitochondrial transmembrane potential (ΔΨm) disruption precedes nuclear chromatin degradation: that monovalent thiol‐reactive compounds inhibit apoptosis induced by diamide, glucocorticoids, irradiation, and topoisomerase inhibition; that the critical thiols determining cell fate after exposure to diamide, glucocorticoids, or DNA damage are likely to be located in the mitochondrial matrix; and that thiol oxidation and reduction are critical for apoptosis induction by some stimuli (glucocorticoids. DNA damage), but not by Fas/CD95 cross‐linking. Taken together, these findings suggest that, at least in some pathways of apoptosis, mitochondrial thiols constitute a critical sensor of the cellular redox potential.
Alterations in intracellular reactive oxygen species generation and redox potential modulate mast cell function
Authors: Wolfreys Karen , Oliveira David B. G.
Published: March 2007

The administration of mercuric chloride (HgCl2), gold compounds, or D‐penicillamine to Brown Norway (BN) rats causes a T helper (Th)2 cell‐associated autoimmune syndrome characterized by the production of a number of autoantibodies, marked elevation of serum IgE concentration, and tissue injury in the form of a vasculitis and arthritis. We have recently shown that the same compounds in vitro sensitize BN rat peritoneal mast cells for IgE‐triggered mediator release and interleukin‐4 mRNA production. We wished to test the hypothesis that these agents influence mast cell function via an effect on intracellular reactive oxygen species (ROS) production/redox balance. Mast cells were obtained from BN rats by peritoneal washout. Incubation with HgCl2, gold compounds or D‐penicillamine (the latter only in the presence of copper ions) led to the intracellular production of ROS as shown by the oxidative production of the fluorescent compound 2′,7′‐dichlorofluorescein. Mast cells were more sensitive than splenocytes to this effect. Direct oxidative stress (exposure to H2O2) produced a similar sensitization for mediator release to that caused by HgCl2. Inhibition of ROS formation by desferrioxamine or catalase diminished the enhancement of IgE‐mediated serotonin release caused by HgCl2, as did replenishment of intracellular glutathione. 2‐Mercaptoethanol exacerbated the toxicity of HgCl2, perhaps due to the formation of a lipophilic complex that enhanced HgCl2 uptake. Blocking of glutathione synthesis increased the toxicity of HgCl2, but also abolished any sensitizing effect on mediator release. These results support three main predictions of our hypothesis: (1) the compounds known to influence mast cell function all lead to the generation of ROS within the mast cell; (2) direct oxidative stress causes sensitization for mediator release by the mast cell; and (3) modulation of ROS production/redox balance within the mast cell modulates the effects of these compounds on mast cell function. The balance of oxidative/antioxidative influences may play an important role in the modulation of mast cell function, particularly in the context of chemically induced autoimmunity.