bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the CD28 costimulatory molecule on subsets of murine intestinal intraepithelial lymphocytes correlates with lineage and responsiveness
Authors: Ohteki Toshiaki , MacDonald H. Robson
Published: March 2007

The CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) αδ+ and γλ+ cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR αβ‐expressing IEL subsets such as CD4+8− and CD4−8α+β+ cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR αβ cells (including CD4−8α+β− and CD4+8αβ+β cells) as well as TcR γλ+ IEL fail to express CD28. Parallel experiments using aged BALB/c‐nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28− cells are considerably larger than CD28+ cells in the gut, although cycling cells were rare in both subsets. Finally CD28− cells in the gut did not proliferate or produce IL‐2 upon stimulation by anti‐CD3 monoclonal antibodies (mAb) and phorbol 12‐myristate 13‐acetate, whereas CD28+ cells in the gut and lymph nodes responded to these stimuli. The response of the CD28+ cells was enhanced by anti‐CD28 mAb. These results suggest that CD28− IEL (CD4‐8α+β− cells, and some CD4+8α+β−cells) may follow a different developmental pathway from that of CD28+ IEL in a thymus‐independent environment, and that expression of CD28 correlates with responsiveness of the cells to triggering via the TcR‐CD3 complex.
Restricted T cell receptor expression by human T cell clones specific for mycobacterial 65‐kDa heat‐shock protein: Selective in vivo expansion of T cells bearing defined receptors
Authors: Henwood Judy , Loveridge Jason , Bell John I. , Gaston John S. Hill
Published: March 2007

We have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65‐kDa heat‐shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigen in vivo.

DR3‐restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF‐derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2+ and Vβ5.6+ clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity‐determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2+ and Vβ5.6+ PB‐derived clones revealed TcR which were identical to those utilized by the SF‐derived clones, showing that the repertoire for recognition of this antigen is stable over time.

Similar studies of TcR expression were carried out on hsp65‐specific, DP4‐restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this defined specificity in the patient's SF.
B cell development in mice with a defective λ5 gene
Authors: Rolink Antonius , Karasuyama Hajime , Grawunder Ulf , Haasner Dirk , Kudo Akira , MeLchers Fritz
Published: March 2007

The surrogate light chain encoded by the two pre‐B cell‐specific genes VpreB and λ5 plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the λ 5gene results in a depletion of B220+ CD43− IgM− pre‐B cells in bone marrow, and in a delayed appearance both of CD5+ as well as CD5− surface immunoglobulin (slg)+ B cells in the periphery. In this report we show that DHJH‐rearranged B220− and B220+, CD43+, c‐kit+, sIgM− pro‐ and pre‐B‐I cells with long‐term capacity to proliferate in vitro on stromal cells in the presence of interleukin‐7 are present in normal numbers in the bone marrow of λ 5T/λ.5T mice at various ages. They express normal levels of VpreB mRNA but, in contrast to normal pre‐B‐I cells, do not express surrogate light chain on their surface. Pre‐B‐I cells from fetal liver and bone marrow of λ 5T/λ 5T mice differentiate with normal kinetics and in normal numbers to sIg+, mitogen‐reactive B cells. These results suggest that the delayed generation of sIg+ B cells in the peripheral, mature compartments of CD5+ and CD5− cells could be accounted for by the daily production of approximately 5 × 105 sIg+ B cells from the pre‐B‐I cell pool in the absence of a normal pool of pre‐B‐II cells.
Functional characterization of the promoter for the human germ‐line T cell receptor J α (TEA) transcript
Authors: De Chasseval Régina , De Villartay Jean‐Pierre
Published: March 2007

TheT cell receptor (TCR)‐α and ‐δ loci are contained on the same chromosomal region, and yet are developmentally and genetically independent. The first element of the Jα cluster (ΨJα) is the site of an active rearrangement in the human thymus (δRec‐ΨJα rearrangement) and is localized downstream of a region expressed as a germ‐line sterile transcript (TEA) in the human developing thymus.We hypothesized that the transcription of TEA could be indicative of (or responsible for) the opening of the Jα to the V(D)J recombinase and undertook to analyze cis‐acting sequences controlling the TEA transcription. The promoter of TEA was characterized. It was part of a region that is highly conserved between human and mouse and contained many sites for the putative binding of T cell‐specific transcription factors. The in vitro activity of this promoter was dependent on the association with an enhancer. A strong DNase I hypersensitive site was found in the vicinity of this promoter again suggesting the possible presence of protein‐DNA interactions in this region. The implications of these results in the general perspective of TCR‐α/δ gene regulation is discussed.
Agents that activate protein kinase C rescue sheep ileal Peyer's patch B cells from apoptosis
Authors: Motyka Bruce , Griebel Philip J. , Reynolds John D.
Published: March 2007

The ileal Peyer's patch (PP) is the major site of B cell production and is a site of immunoglobulin gene diversification in the sheep. Within the ileal PP follicles there is both intense proliferation and death of B cells. We have previously demonstrated that most, if not all of this death can be attributed to apoptosis. Likewise, ileal PP B cells die rapidly by apoptosis in culture ‐ after 6 h many cells appear pyknotic and about 50% of cellular DNA is fragmented. We now show that the DNA fragmentation and cell death of ileal PP B cells can be almost completely abrogated during the first 12 h of culture by the addition of the phorbol esters, phorbol dibutyrate (PBu2) or phorbol myristate acetate. This inhibition of apoptosis could be sustained for greater than 24 h by the concomitant addition of both PBu and the Ca2+ ionophore A23187. However, the rescue of B cells from apoptosis by PBu, with or without Ca2+ ionophore, was prevented by macromolecular synthesis inhibitors or inhibitors of protein kinase C activation. Furthermore, treatment of cultures with PBu, with or without Ca2+ ionophore, resulted in an activated B cell phenotype and a three‐ to fourfold increase in cell proliferation. We conclude that protein kinase C activation in conjunction with an increase in intracellular [Ca2+] can provide the signals necessary to rescue ileal PP B cells from apoptosis, and speculate that these ileal PP B cells are destined to die unless they receive a signal that rescues them from the death pathway.
HLA‐DRβ chain residue 86 controls DRαβ dimer stability
Authors: Verreck Frank A. W. , And Annemarie Termijtelen , Koning Frits
Published: March 2007

Major histocompatibility complex class II molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS‐stable and SDS‐unstable αβ dimers. The ratio of stable vs. unstable αβ dimers varies between murine H‐2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA‐DRB1 and HLA‐DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DRβ chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DRβ chains with a glycine at that position. Reverse‐phase high‐performance liquid chromatography analysis of iodinated peptides, which were eluted from DR dimers with either a DRB1*1101 or a DRB1*1104 β chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides.

The valine/glycine dimorphism is highly conserved, present in most HLA‐DR alleles and influences peptide‐binding. Analysis of the occurrence of theVal86 and the Gly86 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Val86 of Gly86 allelic variant is favored.

Thus, the natural, highly conserved dimorphism at HLA‐DR β chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Val86‐ and Gly86‐containing DR dimers in the presence of SDS.
Induction of CD3δεω) by phorbol 12‐myristate 13‐acetate
Authors: Vangsted Annette , Neisig Anne , Wallin Håkan , Zeuthen Jesper , Geisler Carsten
Published: March 2007

The effect of phorbol 12‐myristate 13‐acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3ω chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Tiα, CD3γ and CD3ζ chains two‐to threefold and the synthesis of Tiβ and CD35δεω complexes five‐ to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti‐CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3ω in retention of TcR complexes. From PMA‐treated CEM cells more than 50‐fold the amount of CD3δεω complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3ω.
T cells specific for the myelin oligodendrocyte glycoprotein mediate an unusual autoimmune inflammatory response in the central nervous system
Authors: Linington Christopher , Berger Thomas , Perry Laura , Weerth Susanne , Hinze‐Selch Dunja , Zhang Yiping , Lu Hsi‐Chi , Lassmann Hans , Wekerle Hartmut
Published: March 2007

Myelin oligodendrocyte glycoprotein (MOG)‐specific T cells mediate an autoimmune inflammatory response in the central nervous system (CNS) that differs radically from conventional models of T cell‐mediated experimental allergic encephalomyelitis (EAE). Using synthetic peptides an encephalitogenic T cell epitope of MOG for the Lewis rat was identified within the extracellular IgG V‐like domain of the protein, amino acids 44‐53 (FSRVVHLYRN).The adoptive transfer of CD4+ T cells specific for this epitope induce an intense, dose‐dependent inflammatory response in the CNS of naive syngeneic recipients. However, unlike the inflammatory response induced by myelin basic protein (MBP)‐specific T cell lines, inflammation mediated by the MOG peptide‐specific T cells failed to induce a gross neurological deficit.

This unexpected observation was not due to a reduction in the overall inflammatory response in the CNS, but was specifically associated with a decrease in the extent of parenchymal (as opposed to perivascular) inflammation, a selective decrease in the number of ED1+ macrophages infiltrating the CNS, and a total lack of peripheral nerve inflammation. The decreased recruitment of macrophages into the CNS could not be ascribed to deficiences in the synthesis of interferon‐γ, tumor necrosis factor‐a, interleukin (IL)‐6 or IL‐2 by the T cell line. Moreover, this sub‐clinical inflammatory response induced severe blood‐brain barrier dysfunction as demonstrated by the induction of severe clinical disease following intravenous injection of a demyelinating MOG‐specific monoclonal antibody. The neurological deficit in EAE thus exhibits an unexpected dependence on the identity of the target autoantigen, which determines the extent and nature of the local inflammatory response and ultimately the extent of the neurological deficit.
The NH2‐terminal domain of rat CD2 binds rat CD48 with a low affinity and binding does not require glycosylation of CD2
Authors: Van Der Merwe P. Anton , McPherson Douglas C. , Brown Marion H. , Barclay A. Neil , Cyster Jason G. , Williams Alan F. , Davis Simon J.
Published: March 2007

CD2, CD48 and CD58 are structurally similar cell adhesion‐molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48‐CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48‐CD4‐coated polystyrene Dynabeads™ formed rosettes on rat CD2‐transfected COS‐7 cells, and this rosetting was blocked by anti‐CD2 (OX34) and anti‐CD48 (OX45) monoclonal antibodies. We used sucrose‐gradient ultracentri‐fugation to show that sCD48‐CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2‐terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48‐CD2 interaction is 4 × 105 M−1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2‐terminal IgSF domain with a low affinity and that binding is independent of glycosylation.
Repeated induction of nitric oxide synthase and leishmanicidal activity in murine macrophages
Authors: Cunha Fernando Q. , Assreuy Jamil , Xu Damo , Charles Ian , Liew Foo Y. , Moncada Salvador
Published: March 2007

Murine macrophages express high levels of nitric oxide (NO) synthase and produce large amounts of NO when stimulated with interferon‐y plus lipopolysaccharide in vitro. The expression of NO synthase peaks at 12 h after stimulation and declines rapidly to the background level by 72 h. These macrophages can be repeatedly reactivated to express similar levels of NO synthase. The reactivation is not due to newly divided cells since peritoneal macrophages which do not divide in vitro and J774 cells cultured in the presence of colchicine can also be restimulated to express NO synthase. The reactivation is accompanied by re‐expression of NO synthase mRNA, as assessed by polymerase chain reaction analysis. Furthermore, the reactivated macrophages are fully capable of killing the intracellular protozoan parasite Leishmania major.
Inhibition of complement alternative pathway in mice with Fab antibody to recombinant adipsin/factor D
Authors: Pascual Manuel , Catana Emmanuelle , White Tyler , Spiegelman Bruce M. , Schifferli Jürg A.
Published: March 2007

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r‐adipsin) were produced in rabbits. Anti‐r‐adipsin IgG was shown to bind to radiolabeled r‐adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of antir‐adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4‐h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti‐r‐adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti‐r‐adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.
In vivo priming effect during various stages of ontogeny of an influenza A virus nucleoprotein peptide
Authors: Kuzu Hiroshi , Kuzu Yukiko , Zaghouani Habib , Bona Constantin
Published: March 2007

In vivo priming of cytotoxic T lymphocytes (CTL) by an influenza virus nucleoprotein (NP) peptide was studied at various stages of development. Adult mice immunized twice with the NP peptide in complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, produce significant CTL responses. Neonates immunized at birth with large amounts of NP peptide and boosted twice with the peptide during adulthood, also mount a weak but significant CTL response. By contrast, offspring from mothers immunized with the NP peptide at days 15, 17, and 19 of pregnancy showed unresponsiveness to the peptide subsequent to a similar regimen of peptide immunization at the age of 1 month. The data indicate that the contact of T cell precursors with antigen during fetal life induces CTL tolerance, whereas, afterbirth the precursors are not susceptible to tolerogenic signals.
The gene loci for immunoglobulin heavy chains in precursor B cell lines from a patient with severe combined immunodeficiency appear able to participate in DNA rearrangement but have a germ‐line configuration
Authors: Kamachi Yoshiro , Ichihara Yoshikazu , Tsuge Ikuya , Abe Tokuichiro , Torii Shinpei , Kurosawa Yoshikazu , Matsuoka Hiroshi
Published: March 2007

In a previous study (Immunogenetics 1988. 27: 330) with Epstein‐Barr virus, we established lines of precursor B cells from bone marrow cells of a patient with severe combined immunodeficiency in whom the numbers of B cells and T cells were markedly reduced. Although based on their surface markers these cell lines appeared to be at an early stage of B cell differentiation, the gene loci for immunoglobulin heavy chains (IgH) retained the germ‐line configuration on both chromosomes in almost all the transformants. In this study, we found that the enhancer sequence, located between the Jh and μ genes, was hypomethylated and an abundance of the germ‐line Cμ transcript was detected in these cell lines by Northern hybridization. These results suggest that the chromatin structure of the IgH gene locus in these cell lines is accessible to VDJ recombinase and is able to participate fully in DNA rearrangement. By contrast, we did not detect transcripts of the RAG‐1 and RAG‐2 genes, which are required for V(D)J recombination at gene loci for immunoglobulin and T cell receptors. Thus, it seems likely that these cell lines fail to initiate the V(D)J recombination process because of some deficiency in the formation of VDJ recombinase, which includes the inability to express RAG genes.
The majority of human tonsillar CD5+ B cells express somatically mutated Vχ4 genes
Authors: Ebeling Saskia B. , Schutte Mieke E. M. , Logtenberg Ton
Published: March 2007

We have fractionated human tonsillar B cells on the basis of CD5 expression and determined the nucleotide sequences of immunoglobin light chain variable (V) regions encoded by the single member of the Vχ4 gene family in both CD5+ and CD5− populations. The majority of cDNA from both CD5+ and CD5− B cells populations harbored somatic mutations. Thus, human tonsillar CD5+ B cells, unlike their murine counterparts, are capable of activating their somatic hypermutation mechanism, resulting in the accumulation of somatic mutation in the VL regions.
The efficient bovine insulin presentation capacity of bone marrow‐derived macrophages activated by granulocyte‐macrophage colony‐stimulating factor correlates with a high level of intracellular reducing thiols
Authors: Frosch Stefanie , Bonifas Ursula , Eck Hans‐Peter , Bockstette Michael , Droege Wulf , Rüde Erwin , Reske‐Kunz Angelika B.
Published: March 2007

Bone marrow‐derived macrophages (BMMΦ) were shown before to function as antigen‐presenting cells. We show here, that the antigen presentation capacity of BMMΦ depends on the nature of the antigen and is differently regulated by the lymphokines interferon‐γ (IFN‐γ) and granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF). When bovine insulin (BI) was employed as antigen, only BMMΦ treated with GM‐CSF (GM‐CSF‐MΦ) were efficient presenters, but when presentation of the antigens ovalbumin and conalbumin was tested, IFN‐γ‐pulsed BMMΦ (IFN‐γ‐M Φ) proved superior to GM‐CSF‐MΦ. The lack of efficient BI presentation function of IFN‐γ‐M Φ was only obvious, when native BI was used as antigen. Preprocessed BI was presented by IFN‐γ‐M Φ with drastically higher efficiency than by GM‐CSF‐M Φ Because processing of insulin depends on reduction of disulfide bonds, we analyzed the content of intracellular reducing thiols within IFN‐γ‐M Φ, GM‐CSF‐M Φ, and untreated BMMΦ. Only after stimulation with GM‐CSF did the amount of reduced glutathione and cysteine strongly increase, while IFN‐γ did not efficiently augment the intracellular content of both thiols. These findings suggest that the lymphokines IFN‐γ and GM‐CSF differently interfere with the processing capacity of BMMΦ by differently regulating the intracellular concentration of the thiols reduced glutathione and cysteine. A high level of these thiols induced by GM‐CSF correlates with a prominent capacity to present the antigen bovine insulin.
New simplified molecular design for functional T cell receptor
Authors: Brocker Thomas , Peter Annick , Traunecker Andre , Karjalainen Klaus
Published: March 2007

We have produced a chimeric single‐chain T cell receptor (TcR) that combines the specific antibody recognition function and TcR/CD3 signaling properties within the same polypeptide chain. This hybrid molecule consisted of a single‐chain antibody combining site that was connected over a short spacer to the transmembrane and cytoplasmic region of CD3ζ. When expressed on TcR− or TcR+ T cell hybridomas it could mediate recognition of relevent target cells and subsequent production of lymphokines; i.e. it could functionally replace the TcR/CD3 complex. Therefore, the single‐chain TcR model presented here represents an interesting and useful means for the creation of T cells with new specificities.
Selective disbalances of peripheral blood T lymphocyte subsets in human CD3γ deficiency
Authors: Timón Marcos , Arnaiz‐Villena Antonio , Rodríguez‐Gallego Carlos , Pérez‐Aciego Paloma , Pacheco Alberto , Regueiro Jose R.
Published: March 2007

The selection of T lymphocytes in the thymus and their activation upon the encounter with foreign antigens in the periphery require the aggregation and signals of the Tcell receptor (TcR)/CD3 complex and several surface molecules termed coreceptors (notably CD4 or CD8 and CD45). The spatial arrangement and interactions of the different molecules in the resulting multimolecular recognition structure are mostly unknown. Here we report, from studies on a healthy human CD3γ deficiency, that the lack of the CD3γ component of the TcR/CD3 complex is associated with a long‐term severe defect of peripheral blood CD4+CD45RA+ and CD8+ lymphocytes, whereas CD4+CD45RO+, B and natural killer lymphocytes are unaffected. These results suggest that the CD3y site of the TcR/CD3 complex is required for the peripheral representation of certain Tcell types.
Evidence that macrophages are programmed to die after activating autologous, cloned, antigen‐specific, CD4+ T cells
Authors: Richardson Bruce C. , Buckmaster Tarek , Keren David F. , Johnson Kent J.
Published: March 2007

The bone marrow produces large numbers of monocytes daily, but the mechanisms balancing production and elimination are unknown. In this report we demonstrate that macrophages (Mϕ) undergo apoptosis after activating autologous CD4+ cells. Since apoptosis is a genetically programmed response, these results argue that MΦ death can be part of a normal immune response. This event may have relevance to monocyte/Mϕ homeostasis, as well as immune response regulation and host defenses to intracellular organisms.
An NF‐χB‐like element plays an essential role in interleukin‐1‐mediated costimulation of the mouse interleukin‐2 promoter
Authors: Strieker Kirstin , Serfling Edgar , Krammer Peter H. , Falk Werner
Published: March 2007

Interleukin‐1 (IL‐1) costimulation is required for efficient IL‐2 synthesis and IL‐2 receptor (IL‐2R) expression of T cells. The molecular events leading to these effects are largely unknown. We utilized an IL‐1‐responsive and an IL‐1‐non‐responsive subclone of the mouse thymoma cell line EL4 to investigate how IL‐1 activates IL‐2 gene expression. We correlated IL‐2 promoter activity with the activity of the endogenous IL‐2 gene, thereby showing the biological significance of our results. Our experiments provide new functional data showing that a major target of IL‐1 mediated costimulation is the χB‐like site, T cell element distal TCEd (GGGATTTCAC), of the IL‐2 promoter. Thus, deletion or mutation of TCEd within a complete IL‐2 promoter abrogated IL‐1 costimulation in the IL‐1 responsive EL4 subclone. Therefore. The TCEd element is functionally essential for the effect of IL‐1.We also identified a nuclear factor (NF), IL‐1 NF, that binds to the TCEd site after IL‐1 stimulation. This factor was only present in the IL‐1‐reponsive EL4 subclone and not in the IL‐1‐non‐responsive subclone after IL‐1 stimulation and did not appear after phytohemagglutinin (PHA)‐treatment. Binding of IL‐1 NF to the TCEd site was competed by a typical χB oligonucleotide, suggesting that it is similar to NF‐χB in its DNA‐binding properties. However, the TCEd element was only activated by costimulation with PHA and IL‐1 whereas a typical χB element was already activated by IL‐1 alone. These data suggest that the biological function of the TCEd element of the IL‐2 promoter differs from that of a canonical χB element. Our data provide new evidence that IL‐1 acts on the IL‐2 promoter by activating the TCEd element via the transcription factor IL‐1 NF. Furthermore, activation of this element requires two signals, delivered by IL‐1 and PHA, in this way reflecting the activation requirement for the endogenous IL‐2 gene.
Chemotaxins inhibit neutrophil adherence to and transmigration across cytokine‐activated endothelium: Correlation to the expression of L‐selectin
Authors: Moser René , Olgiati Licia , Patarroyo Manuel , Fehr Jorg
Published: March 2007

Non‐activated neutrophils strongly adhere to cytokine‐activated human umbilical vein endothelial cells (HUVE). However, activation of neutrophils by different chemotactic mediators led to potent inhibition of this endothelial‐dependent interaction. For different formylated peptides, concentrations leading to maximal adherence inhibition coincided with those known for inducing maximal chemotactic migration of neutrophils. In terms of maximal adherence inhibition, a rank list was found in the order of N‐formyl‐Met‐Leu‐Phe > C5adesArg > interleukin‐8 > C5a ⩾ leukotriene B4, whereas platelet‐activating factor, and lipopolysaccharide showed no inhibition. This rank order was congruent to that of down‐regulation of neutrophil L‐selectin detected by the monoclonal antibody Leu‐8. Moreover, the dose‐dependent increase of neutrophil adherence inhibition corresponded to the loss of L‐selectin expression. Concentrations higher than that required for maximal inhibition led to a dose‐dependent decrease of inhibition, which was accompanied by increasing expression of neutrophil CD11/CD18. In contrast to the capacity of non‐activated neutrophils to migrate across interleukin‐1‐activated HUVE monolayers, transmigration was significantly impaired after chemotactic activation.
CDw50 and ICAM‐3: Two names for the same molecule
Authors: Juan Manel , Vilella Ramon , Mila Jordi , Yagüe Jordi , Miralles Agusti , Campbell Kerry S. , Friedrich R. Joachim , Cambier John , Vives Jordi , De Fougerolles Antonin R. , Springer Timothy A.
Published: March 2007

CDw50 differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDw50 monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC). To understand the function of CDw50 better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (ICAM)‐3, the third Iigand of lymphocyte function‐associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM‐3 and CDw50 are the same glycoprotein, a 120‐kDa surface molecule with presumably an important role in the immune responses.
Activation with superantigens induces programmed death in antigen‐primed CD4+ class II+ major histocompatibility complex T lymphocytes via a CD11a/CD18‐dependent mechanism
Authors: Damle Nitin K. , Leytze Gina , Klussman Kerry , Ledbetter Jeffrey A.
Published: March 2007

Staphylococcal enterotoxin superantigens (SAg) bind class II major histocompatibility complex (MHC) molecules on antigen‐presenting cells (APC) and upon cell‐to‐cell contact stimulate proliferation of T cells expressing appropriate Vβ gene products. In addition, SAg can also deliver negative signals to Ag‐specific T cells resulting in a state of unresponsiveness or a loss of viability. The present study examines the functional consequences of a direct interaction of SAg with alloAg‐specific class II MHC+ CD4+ T cell lines (TCL). Our results demonstrate that SAg induce programmed death (apoptosis) in a majority of Ag‐specific CD4+ T cells accompanied by genomic DNA fragmentation. SAg binding to Ag‐specific TCL resulted in a rapid mobilization of intracellular free calcium ([Ca2+]j) and transcription of a number of cytokine genes including interleukin‐2 (IL‐2), IL‐4, interferon‐γ (IFN‐γ), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and granzyme B indicating the activation of primed T cells. Both SAg‐induced cytokine gene expression as well as subsequent death were significantly inhibited by a tyrosine kinase inhibitor herbimycin A and also by cyclosporin A. SAg‐induced death of primed T cells was also inhibited by monoclonal antibodies (mAb) directed at the CD11a/CD18 molecule but not those reactive with other T cell surface molecules such as CD2, CD7, CD28, CD29 or CD49d. None of these mAb, including anti‐CD11a/CD18, had any effect on SAg‐induced expression of IL‐2 and IL‐4 genes or SAg‐induced [Ca2+]i response. Addition of cytokines such as IL‐1a, IL‐2, IL‐4, IL‐6, GM‐CSF, IFN‐γ, tumor necrosis factor (TNF‐α, or TNF‐β), or neutralizing Ab to these cytokines had no effect on SAg‐induced death of Ag‐specific TCL. The T cells which survived the death‐inducing effects of SAg showed down‐regulation of the CD3/Tcell receptor and up‐regulation of CD2 and HLA‐DR expression, and upon re‐exposure to the same SAg upregulated expression of mRNA for IL‐2 and IFN‐γ. Presentation of SAg by B7+ ICAM‐1+ LFA‐3+ DR+ professional APC was also able to induce the death of Ag‐specific TCL. Together these results suggest that the activation with SAg causes programmed death of Ag‐specific TCL cells via a mechanism that requires late participation of the CD11a/CD18 molecule.
Multiple T cell specificities for Bet v I, the major birch pollen allergen, within single individuals. Studies using specific T cell clones and overlapping peptides
Authors: Ebner Christof , Schenk Siegfried , Szépfalusi Zsolt , Hoffmann Karin , Ferreira Fatima , Willheim Martin , Scheiner Otto , Kraft Dietrich
Published: March 2007

Twenty‐five T cell clones specific for Bet v I were established from the peripheral blood of two birch pollen‐allergic patients. The T cell epitopes of these clones were mapped using dodecapeptides overlapping for 2 amino acids (neighbors share 10 residues) spanning the whole amino acid sequence of the protein (159 amino acids). In total, 7 epitopes could be detected. One donor displayed 6 distinct T cell specificities for the Bet v I molecule in 14 T cell clones; for the other donor, 4 stimulating peptides for 11 clones could be identified. Two T cell epitopes were recognized by both subjects. One of these might represent an immunodominant epitope located at amino acid position 77–92 of the Bet v I molecule, as in 13/25 T cell clones activation could be induced by this amino acid sequence. One T cell clone reacted with purified pollen‐derived Bet v I, but neither with any peptide synthesized according to a Bet v I‐encoding cDNA nor with the respective recombinant protein.

Upon stimulation with allergen, the majority of the clones (21/24) revealed the THO or TH2 type of cytokine production (interleukin‐4 production), indicating their importance in the pathogenesis of the allergic disease.
Immunization of mice with the N‐terminal (1–272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes
Authors: Schirmbeck Reinhold , Zerrahn Jens , Kuhröber Andreas , Deppert Wolfgang , Reimann Jörg
Published: March 2007

Immunization of C57BL/6 (B6) mice (H‐2b) with the “large tumor antigen” (T‐Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8+ cytotoxic T lymphocytes (CTL) in vivo. CD8+ CTL primed in vivo by this non‐structural 708‐amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H‐2b target cells, either transfected with an SV40 T‐Ag‐encoding vector, or transformed by SV40 infection. H‐2b RMA‐S transfectants expressing the complete 708 aa T‐Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N‐terminal 272 aa fragment of the T‐Ag. Hence, this fragment contains the structure (s) required for a soluble protein to enter the “endogenous” class I‐restricted antigen processing and presentation pathway for CD8+ CTL activation. In soluble form, the complete T‐Ag or the N‐terminal T‐Ag fragment sensitized in vitro RBL5 cells for lysis by T‐Ag‐specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapetides (derived from N‐terminal T‐Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T‐Ag and its 272‐aa N‐terminal fragment can enter the “endogenous” processing pathway and prime CD8+ CTL in vivo and in vitro.
T cell activation by a Trypanosoma brucei brucei‐deriyed lymphocyte triggering factor is dependent on tyrosine protein kinases but not on protein kinase C and A
Authors: Bakhiet Moiz , Mix Eilhard , Kristensson Krister , Wigzell Hans , Olsson Tomas
Published: March 2007

Trypanosoma brucei brucei releases a lymphocyte‐triggering factor (TLTF) that activates CD8+ T cells. We here study second messenger mechanisms in this activation, i. e. the effects of protein kinase C (PKC), protein kinase A (PKA) and tyrosine kinases (TPK) inhibitors on TLTF‐induced interferon‐γ (IFN‐γ) secretion and proliferation in lymphoid cell cultures. The effects were compared to those obtained by phytohemagglutinin (PHA) stimulation. Rat spleen mononuclear cells (MNC) and spleen MNC from a mutant mouse strain possessing CD8+ T cells but lacking CD4+ T cells were used as responder cells.

Although both the PKC and the PKA inhibitors suppressed PHA‐induced IFN‐γ secretion and proliferation of rat MNC and mouse CD8+CD4− MNC, they had no effect on the same TLTF‐induced responses. The TPK inhibitor genistein, however, strongly suppressed TLTF‐induced activation of both types of responder cells to IFN‐γ secretion and the TLTF‐induced proliferation of mouse CD8+CD4− MNC. The suppressive effects of the drugs could be overcome by ionomycin and tetradecanoylphorbol acetate, which show that the effects were not due to drug nonspecific cellular toxicity of the drugs. We conclude that TLTF activates CD8+ T cells through pathways other than the PKC‐ or PKA‐dependent signal transduction, and that TPK may be involved in the triggering.
Lysis of infected cells in vivo by antiviral cytolytic T cells demonstrated by release of cell internal viral proteins
Authors: Kyburz Diego , Speiser Daniel E. , Battegay Manuel , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

Immunocompetent adult mice infected with lymphocytic choriomeningitis virus (LCMV) generate a strong antiviral cytotoxic T cell response that clears virus from all organs. Although there is good evidence that immune cytotoxic T lymphocytes (CTL) kill target cells in vitro, in vivo it is debated whether antiviral activity of CD8+ T cells is mediated via direct target cell lysis or via soluble mediators.

To demonstrate CD8+ T cell‐mediated destruction of infected cells in vivo a specific cell‐internal releasable marker was used as label, i.e. the nucleoprotein (NP) of LCMV. Since LCMV is non‐cytopathic the viral NP will only be released in substantial amounts because of destruction of infected host cells by immune CTL. It is shown here that the amount of NP released from infected and 51Cr‐labeled target cells in vitro correlated well with the amount of radioactivity released. Viral NP released in vivo by CTL is bound and masked by the anti‐NP antibodies that are produced very early and efficiently. However, NP could readily be detected in sera of LCMV‐infected CD8+ competent mice that could not generate antibodies specific for the NP because they were treated with a depleting anti‐CD4 antibody. NP was also detected in the cerebrospinal fluid of mice suffering from CD8+ T cell‐mediated lymphocytic choriomeningitis after intracerebral infection. NP titers in sera of anti‐CD4‐treated LCMV‐infected mice exhibited a peak around day 7–8 when CTL activity was highest. When mice were CD8 T cell‐depleted with anti‐CD8 monoclonal antibody or in LCMV‐carrier mice, no NP was detected in the serum. Highly activated LCMV‐specific CTL adoptively transfused to LCMV‐infected irradiated recipient mice also caused a time‐dependent release of NP into serum. This confirms that the CD8+ population is responsible for the release of NP from infected host cells. These results represent an in vivo correlate of CTL‐mediated cytolysis and evidence that antiviral cytotoxic T cells are cytolytic in vivo. They also suggest that antibody responses to internal antigens of non‐cytopathic viruses may signal CD8+ Tcell‐mediated destruction of infected host cells.
The extended hinge region of IgG3 is not required for high phagocytic capacity mediated by Fcγ receptors, but the heavy chains must be disulfide bonded
Authors: Aase Audun , Sandlie Inger , Norderhaug Lars , Brekke Ole Henrik , Michaelsen Terje E.
Published: March 2007

Feγ receptor (FcγR) phagocytosis and respiratory burst were induced by chimeric mouse‐human anti‐(4‐hydroxy‐5‐iodo‐3‐nitrophenyl) acetyl IgG3 antibodies with mutations in hinge and/or in CH1 region. IgG3 mutants with different hinge length ranging from 47 to 0 amino acids, an IgG3 molecule with an artificial hinge of just one cysteine residue (HM‐1), and two hybrid IgG3 molecules with IgG4 hinge or IgG4 CH1‐hinge were tested. Using the monocytic cell line U937 as effector cells, the mutated IgG3 molecules were very similar, revealing high activity, while the IgG3/IgG4 hybrids revealed a slightly reduced activity. However, the hingeless (0‐h) mutant was negative, except after interferon‐γ stimulation when it became slightly positive. Interestingly, HM‐1 was as active as the IgG3 mutants. With polymorphonuclear leucocytes (PMN) as effector cells we obtained some day‐to‐day variations, but all the IgG3 mutants were highly active, with the two shortest hinge mutants somewhat less active The IgG3/IgG4 hybrid molecules revealed an intermediate activity, while IgG4 wild‐type and the 0‐h mutant were negative. However, the HM‐1 molecule revealed an activity similar to that of the IgG3 mutants. The phagocytic activity of U937 was inhibited by monomeric IgG, indicating the importance of FcγR III. In contrast, with PMN both blockage of FcγRII and cleavage of FcγRIII were required to significantly reduce the phagocytosis and respiratory burst, thus showing that both receptors contribute to the effect. These results demonstrate that the extended IgG3 hinge region is not necessary for a high phagocytic activity and that the major structural importance of the hinge is to connect the two heavy chains in this region.
Propriocidal apoptosis of mature T lymphocytes occurs at S phase of the cell cycle
Authors: Boehme Stefen A. , Lenardo Michael J.
Published: March 2007

We found that mature nontransformed CD4+ and CD8+ T lymphocytes could be made susceptible to T cell receptor(TcR)‐mediated apoptosis by pretreatment with interleukin‐4 (IL‐4) or interleukin‐2 (IL‐2). The degree of susceptibility to death could be correlated with the level of cell cycling as measured by thymidine incorporation, cell doubling times, or the number of cells incorporating bromodeoxyuridine during S phase. However, using pharmacologic cell cycle blocking agents, we found that progression through the cell cycle was not required for cell death. Rather, we found that cells must be in a certain phase of the cell cycle to be susceptible to TcR‐mediated death. Cells blocked in G1 phase were resistant to T cell receptor‐induced apoptosis, whereas cells blocked in S phase were susceptible. These observations suggest that an important feature of growth lymphokines is their ability to drive T cells into portions of the cell cycle where they are sensitive to antigen receptor‐induced apoptosis. Furthermore, these results provide additional evidence that the T cell growth lymphokines IL‐2 and IL‐4 may participate in the down‐regulation of T cell responses by apoptosis a pathway we have termed “propriocidal regulation”.
Nocardia fractions, NLD and NWSM, induce tumor necrosis factor‐α secretion in human monocytes: Role of protein kinase C
Authors: Mege Jean Louis , Jacob Thiery , Bongrand Pierre , Capo Christian , Myssiakine Evguenine B. , Barot‐Ciorbaru Rita
Published: March 2007

Nocardia lysozyme digest (NLD) and Nocardia water‐soluble mitogen (NWSM) are two fractions derived from Nocardia opaca. In this report, we demonstrated that both fractions elicited significant secretion of tumor necrosis factor‐α (TNF‐α) in human monocytes. Supernatants from monocytes stimulated with NWSM and low concentrations of NLD displayed a cytotoxic activity against TNF‐α‐sensitive L929 cells, but supernatants from monocytes stimulated with high concentrations of NLD failed to lyse L929 cells. This latter phenomenon might be related to the secretion of an inactive form of TNF‐α or the release of an inhibitor of TNF‐α cytotoxic activity. Since it is well established that protein kinase C (PKC) plays a major role in the signaling of several monocyte activators, we investigated the putative role of PKC in cytokine synthesis induced by NLD and NWSM fractions. TNF‐α secretion in response to both Nocardia fractions was inhibited by sphingosine, staurosporine and calphostin C, known PKC inhibitors, as well as by a PKC depletion procedure. In addition, NLD and NWSM induced a transient increase in [3H] phorbol dibutyrate binding, which assessed the activation of PKC. The data suggest the involvement of PKC in the signaling of NLD and NWSM fractions leading to the synthesis and the secretion of TNF‐α by human monocytes.
Resistance to collagen‐induced arthritis in rats and rhesus monkeys after immunization with attenuated type II collagen
Authors: 't Hart Bert A. , Bakker Nicolaas P. M. , Jonker Margreet , Bontrop Ronald E.
Published: March 2007

Immunization of susceptible rodent or primate species with type II collagen (b‐CII) from bovine origin induces type II collagen‐induced arthritis (CIA). The disease is characterized as a systemic polyarthritis associated with humoral and cellular autoimmunity to CII and shares similarity with human arthritic diseases. The objective of this study was to develop a procedure for induction of resistance to CIA in animals, which possess a certain major histocompatibility complex phenotype that makes them prone to develop CIA (susceptible). It is shown that by immunization with an attenuated form of CII, in which arthritogenic epitopes have been destroyed by heat denaturation, disease resistance is induced in a susceptible inbred rat strain (RT‐1U) and in an outbred population of susceptible rhesus monkeys (lacking the Mamu‐A26 allele). In both species the disease resistance is connected with modulation of anti‐CII autoantibodies of the IgM isotype. This protocol may provide a basis for effective and safe methods to induce protection to autoimmune arthritis in those subjects that are genetically prone to develop such a disease.
Analysis of intraepithelial lymphocytes from major histocompatibility complex (MHC)‐deficient mice: No evidence for a role of MHC class II antigens in the positive selection of Vδ4+γδ T cells
Authors: Schleussner Cathrin , Ceredig Rhodri
Published: March 2007

Three‐color flow cytometric analysis was carried out with intraepithelial lymphocytes from mice deficient in expression of major histocompatibility complex (MHC) antigens. These experiments were done to address the possible role of MHC class II molecules in the positive selection of Vδ4+ γδ T cells. By analyzing mice deficient MHC class II antigens alone or in combination with MHC class I antigens, no evidence was found for positive selection of Vδ4+ cells among CD8a+ or CD4−CD8− subpopulations of γδ T cell receptor‐positive cells. Because V54+, CD8a+ cells were reported to be positively selected on I‐Ek and hybrid I‐Ek/b molecules, class II‐deficient animals were crossed with I‐Ek transgenic mice and progeny examined for Vδ4 expression. Again, no evidence for positive selection was found. Interestingly, in MHC class I‐deficient animals, the total number of γδ T cells was about twofold higher than in control and MHC class II‐deficient mice and the proportion of V8δ‐expressing cells was correspondingly decreased. Taken together, these results cast doubt on a major role for conventional MHC antigens in shaping the γδ T cell repertoire of intraepithelial lymphocytes.