bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies raised against peptide fragments of bovine αs1‐casein cross‐react with the native protein, but recognize sites distinct from the determinants on the protein
Authors: Shon Dong‐Hwa , Enomoto Atsushi , Yamauchi Kunio , Kaminogawa Shuichi
Published: March 2007

Bovine αs1‐casein (αs1‐CN) and its peptides 61–110 and 91–110, which contain both T and B cell determinants on αs1‐CN and can elicit peptide‐native protein cross‐reactive antibodies, were selected as model antigens to study whether or not the immune response to the peptides is similar to that to the corresponding regions of the native protein, because they both have a similar disordered conformation in solution. Both αs1‐CN‐ and peptide 61–110‐primed T cells responded to peptides 61–80 and 91–100, but not to peptides 76–95 and 101–110. In addition, T cells immunized with peptide 91–110 were also stimulated by peptide 91–100, but not by peptide 101–110. These results suggest that the location of the T cell determinant was almost the same in αs1‐CN and its peptides. On the contrary, antibodies raised against αs1‐CN bound to peptides 76–95 and 91–100, but not to peptides 61–80 nor 101–110, while anti‐peptide 61–110 antibodies preferentially reacted with peptides 61–80 and 101–110, and anti‐peptide 91–110 antibodies also bound to peptide 101–110 but not to peptide 91–100. These results indicate that the B cell epitopes were not similar between αs1‐CN and its peptides. This difference may have arisen because the antigen‐B cell or T‐B interactions required for the development of a specific antibody response occurred in a different manner between αs1‐CN and its peptides. These findings may be useful for basic studies on immunology, and could also be applied to the design of new peptide vaccines.
Antibody responses to a synthetic peptide‐based malaria vaccine candidate: influence of sequence variants of the peptide
Authors: Etlinger Howard M. , Renia Laurent , Matile Hugues , Manneberg Michael , Mazier Dominique , Trzeciak Arnold , Gillessen Dieter
Published: March 2007

A synthetic peptide (Asn‐Ala‐Asn‐Pro)3, representing a protective sequence from the sporozoite stage of Plasmodium falciparum, conjugated to tetanus toxoid has undergone clinical testing. Although some protection was obtained, anti‐parasite responses were generally low. In attempting to improve the anti‐parasite protein antibody response, we evaluated the efficacy of tetanus toxoid conjugates containing seven sequence variants of the peptide. Most of the conjugates tested in both mice and monkeys elicited anti‐peptide antibodies with fine specificity differences, although there was a broad degree of cross‐reactivity. In general, each conjugate tested evoked similarly high anti‐sporozoite antibody responses in both species and, thus, based on antibody titer no evidence for a superior vaccine candidate was obtained. The biological activity of one antiserum actually increased the penetration of sporozoites into human liver cells. In contrast, antisera against the other conjugates inhibited sporozoite penetration, but to a similar degree. Based on these two criteria, none of the other conjugates would appear to be better vaccine candidates than the original conjugate. The lack of concordance between anti‐peptide or anti‐sporozoite titers and inhibitory activity in the case of one antiserum indicates that these two measures of antibody cannot always be used for predicting anti‐sporozoite activity.
Vλ and Jλ‐Cλ gene segments of the human immunoglobulin λ light chain locus are separated by 14 kb and rearrange by a deletion mechanism
Authors: Combriato Gabriele , Klobeck H.‐Gustav
Published: March 2007

We have cloned a region of 124 kb of the human immunoblogulin λ light chain locus on chromosome 22 encompassing seven Vλ and seven J‐Cλ gene segments. No further Cλ gene segment was found in a region of 35 kb downstream of Cλ7, which encodes the Ke+Oz− isotype. The Cλ proximal Vλ gene segment VλIII. 1 is located 14.5 kb upstream of Cλ1. The five sequenced Vλ genes have the same transcriptional orientation as the J‐Cλ gene segments which is likely to be true for the majority of the Vλ gene segments in the human λ locus and which suggests a deletion mechanism for DNA rearrangement. This is supported by hybridization of Vλ gene probes to germ‐line and rearranged DNA from λ light chain‐producing cell lines. Sequences of 23 cDNA clones allow to establish a Vλ subgroup classification based on nucleic acid sequence data and an estimate of the J‐Cλ usage.
Genotype‐restricted lymphoproliferation in autoimmune lpr mice
Authors: Matsuzawa Akio , Kimura Mikio , Muraiso Tomoshige , Kominami Ryo , Katagiri Takuya
Published: March 2007

Transfer of bone marrow (BM) from autoimmunity‐prone mice homozygous for the new lymphoproliferation mutation (lprcg) caused systemic lymphoproliferation in irradiated lprcg/lprcg recipients but not in irradiated +/+ recipient (J. Exp. Med. 1990. 171: 519; Eur. J. Immunol. 1991. 21: 63). It was thus hypothesized that the lprcg gene expresses its function at lymph nodes (LN) to provide anomalous lprcg/lprcg lymphoid cells with the environment where they can accumulate. This was confirmed by LN transplantation and BM transfer studies. In the LN transplantation study lpcg/lprcg LN grafts with or without in vitro irradiation swelled and lprcg/+ LN grafts were slightly hyperplastic or apparently normal; however, whereas +/+ LN grafts atrophied in lprcg/lprcg recipients, they were all histologically normal in +/+ and lprcg/+ recipients. Irradiation of lprcg/lprccg LN grafts significantly retarded their swelling in lprcg/lprcg recipients. In the BM transfer study lprcg/lprcg BM cells caused systemic lymphoproliferation in lpr/lpr and gld/+, lprcg/+ recipients and sporadic LN swelling in lprcg/+ recipients but LN atrophy in gld/gld recipients. In the study using both techniques in combination, lpr/lpr LN grafts swelled but gld/gld LN grafts atrophied in lprcg/lprcg BM → +/+ chimeras. All the swollen LN contained Thy‐1+CD4−CD8− lymphoid cells or “double‐negative (DN)” T cells characteristic of the lpr disease. Analysis of DNA restriction fragment length polymorphism demonstrated that lprcg/lprcg DN cells derived from lprcg/lprcg BM cells accumulated in lpr/lpr LN and gld/+, lprcg+ LN. The following conclusions have been drawn: (a) the lprcg gene determines the ability of lprcg/lprcg DN cell to accumulate in LN; (b) this genetic trait is not totally recessive differing from lymphoproliferation; (c) lpr/lpr LN and gld/+, lprcg/+ LN are equivalent to lprcg/lprcg LN in the receptivity of lprcg/lprcg DN cell accumulation thus supporting the allelism of lpr with lprcg and the complementation between gld and lprcg (J. Exp. Med. 1990. 171: 519), respectively; (d) the ability of lprcg/lprcg LN to accumulate DN cells is partially resistant to irradiation; (e) lprcg/lprcg DN cells may cause atrophy of gld/gld LN and +/+ LN and (f) the gld and lpr genes are different from each other in the phenotype expressed at the LN site.
Antigen‐independent T cell activation mediated by a very late activation antigen‐like extracellular matrix receptor
Authors: Takahashi Kazuhisa , Nakamura Tetsuya , Adachi Hiroyasu , Yagita Hideo , Okumura Ko
Published: March 2007

Cell‐cell and cell‐extracellular matrix (ECM) binding mediated by integrin molecules has been implicated in lymphocyte migration and adhesion. We describe here that ECM binding triggers antigen‐independent activation of T cell functions. Fibronectin and vitronectin, when coated on plates, not only acted synergistically on anti‐CD3‐induced serine esterase release in a murine cytotoxic T lymphocyte clone and interleukin 2 production in a murine helper T cell hybridoma but also could trigger these responses alone. All these stimulatory effects of ECM were abrogated by a monoclonal antibody which reacts with a unique very late activation antigen‐like integrin and this monoclonal antibody, when coated on plates, exhibited a similar synergistic effect to that of ECM proteins. Therefore, ECM receptors expressed on activated T cells appear to play an important role in triggering T cell effector functions in localized tissues abundant in ECM proteins.
Preferential expression of Vβ6.7 domain on human peripheral CD4+ T cells. Implication for positive selection of T cells in man
Authors: Cossarizza Andrea , Kahan Melvyn , Ortolani Claudio , Franceschi Claudio , Londei Marco
Published: March 2007

The peripheral T cell receptor repertoire is mainly controlled by the processes of positive and negative selection occurring in the thymus. Studies in normal or transgenic mice have provided compelling evidence for both negative and positive selection. Negative selection is characterized by partial or total disappearance from the periphery of T cells expressing certain Cβ regions, which are normally present in the thymus. Positive selection is chiefly characterized, in the periphery, by an imbalanced ratio between CD4+ and CD8+ T cells expressing a given V domain.

To date little information concerning positive and negative selection has been available in man. We studied the distribution of 4 Vβ domains on CD4+ and CD8+ peripheral T cells of 34 healthy individuals with a wide age‐range (0–96 years). One of the Vβ domain studied, Vβ6.7, is preferentially expressed on CD4+ compared to CD8+ T cells (p > 0.001). No significant differences were observed using the other Vβ domain‐specific monoclonal antibodies (Vβ5.2–5.3, 8 or 12). These results provide evidence that a process of thymic education, similar to that described in murine animal models, may also take place in man.
T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: Evidence for a protein kinase C‐independent staurosporine‐sensitive step
Authors: Boyer Claude , Auphan Nathalie , Luton Frédéric , Malburet Jean‐Marc , Barad Marc , Bizozzero Jean‐Pierre , Reggio Hubert , Schmitt‐Verhulst Anne‐Marie
Published: March 2007

The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti‐TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12‐myristate 13‐acetate (PMA). After binding of the anti‐TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti‐TcR mAb. PMA‐induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC‐depleted cells or in the presence of the PKC inhibitor staurosporine. Anti‐TcR mAb‐induced internalization was maintained in PKC‐depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti‐TcR mAb Fab fragment is non‐stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC‐depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA‐induced phosphorylation of γ and ζ subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross‐linking of the TcR via plastic‐coated anti‐TcR mAb led to phosphorylation of CD3 γ and ϵ and also of ζ, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti‐TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.
Cytokine interactions in experimental cutaneous leishmaniasis. II. Endogenous tumor necrosis factor‐α production by macrophages is induced by the synergistic action of interferon (IFN)‐γ and interleukin (IL) 4 and accounts for the antiparasitic effect mediated by IFN‐γ and IL 4
Authors: Stenger Steffen , Solbach Werner , Röllinghoff Martin , Bogdan Christian
Published: March 2007

Tumor necrosis factor‐alpha (TNF‐α) strongly activates murine peritoneal macrophages (MΦ) for killing of amastigotes from Leishmania major in the presence of low amounts of interferon‐γ (IFN‐γ). Recently, we found that IFN‐γ and interleukin 4 (IL 4) also synergistically enhance the antileishmanial potential of MΦ. In this report, evidence is provided that the synergism of IFN‐γ and IL 4 is based on the ability of the lymphokines to induce the endogenous production of TNF‐α. First, both IFN‐γ and IL 4 as single agents and in combination were potent inducers of TNF‐α production by MΦ infected with L. major amastigotes. Second, the synergistic effect of IFN‐γ and IL 4 on parasite killing by MΦ strongly correlated with their synergistic effect on the release of TNF‐α. Third, the IFN‐γ/IL 4‐mediated parasite elimination was completely abrogated not only in the presence of antibodies to IFN‐γ and IL 4, but also with an antibody specific for TNF‐α. Consistent with the conclusion that endogenously produced TNF‐α accounts for the synergism of IL 4 with IFN‐γ is the finding that Nω‐monomethyl‐L‐arginine, an inhibitor of the L‐arginine‐dependent generation of microbicidal nitrogen intermediates, totally blocked the MΦ activation induced by IFN‐γ combined with IL 4 as well as by IFN‐γ combined with TNF‐α. These results underline the complex interplay of cytokines derived from lymphocytes and MΦ and the role of TNF‐α as pivotal factor for the induction of antileishmanial effector functions.
Ontogeny of the response of mouse thymocytes to interleukin 1 and interleukin 2
Authors: Liberman Isabelle , Drapier Anne‐Marie , Marche Patrice N. , Cazenave Pierre‐André , Rueff‐Juy Dominique
Published: March 2007

In the present report we demonstrate that the in vitro proliferative response of the newborn thymocytes to interleukin (IL) 1 and IL 2, which is remarkably stronger than the adult thymocyte response, is associated with a considerable increase of CD4−CD8− cells expressing a γ/δ T cell receptor (TcR). By polymerase chain reaction analysis we show that the Vγ gene segment usage in the adult and newborn responding cells reflects the developmentally regulated expression of the Vγ gene segments, suggesting that the increase in TcR γ/δ+ cells results from the polyclonal expansion of pre‐existing clones. Surprisingly, although the fetal thymocyte populations contain higher numbers of TcR γ/δ+ cells than the adult and newborn ones, the highest proliferative response to IL 1 and IL 2 is obtained with the newborn thymocytes. Non mutually exclusive hypotheses are discussed to explain these results.
Variable region genes of anti‐histone autoantibodies from a MRL/Mp‐lpr/lpr mouse
Authors: Monestier Marc
Published: March 2007

The variable region nucleotide sequences of seven (five IgM and two IgG) anti‐histone monoclonal antibodies from a single MRL/Mp‐lpr/lpr mouse have been determined. These antibodies are not clonally related and used diverse V, D and J genes. However, six of the seven antibodies have VH segments encoded by genes from the J558 family, two of these (an IgM and an IgG) share an identical VH gene. The isoelectric points of MRA3 and MRA12, the two IgG antibodies of the panel, range from 6.3 to 7.0 and from 6.0 to 6.3, respectively. The second conplementarity‐determining region (CDR) of the VH gene of MRA12 (the most acidic and the most strongly histone‐reactive antibody) includes only two positively charged but five negatively charged amino acid residues. This feature is unusual since the equivalent CDR in most VHJ558 genes are not comprised predominantly of acidic residues and suggests that such negatively charged residues are important for antibody binding to histones.
Predominant appearance of γ/δ T lymphocytes in the liver of mice after birth
Authors: Ohteki Toshiaki , Abo Toru , Seki Shuhji , Kobata Tetsuji , Yagita Hideo , Okumura Ko , Kumagai Katsuo
Published: March 2007

γ/δ T lymphocytes residing in the liver of mice were systematically characterized with respect to their age‐related variation, phenotype and V gene segment usage of γ/δ T cell receptor (TcR). Previous human and murine studies have shown that a high proportion of γ/δ T cells reside in the liver and that such liver γ/δ T cells have lymphoblastic morphology and can spontaneously proliferate in vitro. In the present study, a predominant appearance of γ/δ T cells (up to 23% among CD3+ cells) in the liver was confirmed in 4‐week old mice of various strains. γ/δ T cells in the liver preferentially co‐expressed CD8 antigens, whereas the vast majority of γ/δ T cells in the spleen lacked the CD8 antigens. The identification of γ/δ T cells in various lymphoid and non‐lymphoid organs also revealed the liver to be one of the organs where γ/δ T cell are most abundant. The level of such liver γ/δ T cells showed a clear age‐related variation. In the fetal stage and just after birth, γ/δ T cells were not detectable in the liver (< 0.2%). However, a significantly higher percentage of γ/δ T cells among both the total population of mononuclear cells and CD3+ cells was detected in the liver of young 2‐ to 8‐week‐old mice; this percentage subsequently declined. As the total number of liver mononuclear cells increased in aged mice, the absolute number of liver γ/δ T cells also increased as a function of age. V gene segment usage analysis by the polymerase chain reaction method demonstrated that Vγ1 or Vγ2/Vδ6 were preferentially used by liver γ/δ T cells. The age‐related increase of γ/δ T cells was more prominent in the liver of athymic nude mice, and such γ/δ T cells highly co‐expressed the CD8 antigens and also utilized the Vγ1 or Vγ2/Vδ6 for γ/δ TcR. The predominant appearance of unique γ/δ T cells in the liver, which was inversely related to the existence of the thymus, indicates that these γ/δ T cells may differentiate extrathymically in the liver.
Induction of hyporesponsiveness to an early post‐binding effect of tumor necrosis factor by tumor necrosis factor itself and interleukin 1
Authors: König Mechthild , Wallach David , Resch Klaus , Holtmann Helmut
Published: March 2007

Modulation of cellular responsiveness to tumor necrosis factor (TNF) was studied in the human SV‐80 cells. A marked cytocidal effect is exhibited by these cells at about 4 to 8 h after application of TNF together with protein synthesis inhibitors. Sensitivity of the cells to TNF toxicity was shown to be markedly decreased following their pretreatment with TNF itself or with interleukin (IL) 1 in the absence of protein synthesis inhibitors. The SV‐80 cells respond to TNF also with enhanced phosphorylation of the small heat‐shock protein, HSP27. This TNF effect is much more rapid than the cytocidal effect; it is observed within minutes of TNF application. The response to this effect, just like the response to the cytocidal effect, is markedly decreased following preexposure of the cells to either TNF or IL 1. Responsiveness to both effects of TNF is regained at the same time, about 15 to 20 h following removal of TNF or IL 1. The decrease in responsiveness after pretreatment with TNF or IL 1 does not reflect an inability of the pretreated cells to bind TNF. Although there is an initial decrease in TNF binding after such pretreatment, it is fully reversed already about 5 h following removal of the cytokines. The rate of uptake of TNF by the pretreated cells is also normal. In view of the rapidity of the effect of TNF on the phosphorylation of HSP27, it seems likely that the observed hyporesponsiveness reflects impairment of an early step in a signaling pathway, perhaps common to both the stimulation of phosphorylation and the induction of cell death by TNF. By restricting the duration of the effects of TNF this desensitization mechanism may safeguard against harmful consequences of these effects.
Protective effect of cyclosporin A on immune abnormalities observed in the murine acquired immunodeficiency syndrome
Authors: Cerny Andreas , Merino Ramon , Makino Masahiko , Waldvogel Francis A. , Morse Herbert C. , Izui Shozo
Published: March 2007

The murine leukemia viruses (MuLV) designated LP‐BM5 induce an immuno‐deficiency disease in susceptible strains of mice with many features in common to human acquired immunodeficiency syndrome (AIDS), including lymphadenopathy and profound immunodeficiency associated with enhanced susceptibility to infection and terminal B cell lymphomas. The disease, termed murine AIDS (MAIDS), crucially depends on the presence of B cells and CD4+ T cells, suggesting that mutual activation of these two cell types is central in the pathogenesis of the immunodeficiency syndrome. Cyclosporin A (CsA), whose immunosuppressive effect is attributed mainly to inhibition of interleukin 2 and interferon‐γ expression, interferes in T‐B cell interactions. Here we show that chronic treatment with CsA (40 or 60 mg/kg/day) before and after infection with LP‐BM5 MuLV protects against the development of immunodeficiency disease as assessed by functional, serological and histopathological criteria. The protection was not complete, suggesting both CsA‐sensitive and CsA‐resistant components to the pathogenesis of this syndrome, and was found to be independent of ecotropic MuLV expression. These results underline immunopathological mechanisms in the progression of immune abnormalities in MAIDS that are susceptible to inhibition of CsA and may serve as an experimental basis for developing a treatment of the human disorder with immunomodulators.
Resistance to collagen‐induced arthritis in Biozzi mice is not associated with T cell receptor Vβ gene polymorphism
Authors: Vidard Laurent , Roger Thierry , Bouvet Jean Pierre , Couderc Jacques , Seman Michel
Published: March 2007

H‐I and H‐II high antibody‐responder Biozzi mice, which express the H‐2q permissive haplotype, were shown to be sensitive and refractory to collagen‐induced arthritis, respectively. To assess a possible role of T cell receptor (TcR) Vβ gene deletion or polymorphism in the resistance to arthritis in H‐II mice, the germinal structure of TcR Vβ genes and their expression at the membrane level were compared in both lines. In contrast to H‐2q refractory SWR mice, which exhibit a deletion of about 50% of TcR Vβ gene segments, H‐I and H‐II lines have an identical and complete set of Vβ genes and exhibit no difference in the average expression of Vβ6, Vβ8 and Vβ11 gene products on T cell surface. These results indicate that mechanisms other than Vβ gene deletion or polymorphism can be involved in the resistance of H‐2q‐positive mice to experimental arthritis.
Alternative complement pathway‐mediated myeloid cell cytotoxicity: Repertoire of membrane factors participating in regulation of C3 deposition and cytolysis
Authors: Matsumoto Misako , Sugita Yuji , Seya Tsukasa
Published: March 2007

Most human nucleated cells and cell lines possess C3 step regulators, decayaccelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) and an inhibitor of membrane attack complex (MAC) formation (p18; CD59). Unless DAF and MCP were simultaneously blocked by their antibodies, Mg2+‐EGTA‐human serum treatment did not induce C3 deposition on most nucleated cells. Furthermore, < 20% lysis occurred even after the block of all the three factors. In contrast, three myeloid cell lines, U‐937, HL‐60 and p39, were found to exhibit unusual C3 deposition or cytolysis. U‐937 possessed DAF and MCP but lacked p18, and about 50% was lysed by treatment with anti‐DAF and anti‐MCP followed by Mg2+‐EGTA‐serum, which caused C3, C5 and C8 deposition. Anti‐DAF evoked similar but less complement (C) deposition and cytolysis while anti‐MCP alone did not, although it enhanced the anti‐DAF‐mediated C deposition and cytolysis. Thus, once the C3 step is overcome, U‐937 is attacked by the late components leading to cytolysis because of the absence of p18. On the other hand, HL60 allowed the deposition of C3 by blocking of either DAF or MCP followed by the Mg2+‐EGTA‐serum treatment. C5, C8 and C9 were subsequently deposited but resulted in no lysis. Lysis of 60% was attained by the additional blocking of p18. Thus, HL60 is poorly protected by C3 and C9 step regulation. Strikingly, extensive C3 deposition occurs on p39 without any antibody treatment, suggestive of the presence of unique alternative pathway activators. However, little cytolysis was induced on p39 even by blocking of all three inhibitors with antibodies. These results suggest that in activation of the alternative pathway on myeloid cells, C3 step is controlled by the inhibitors and alternative pathway activators, and C‐mediated cytolysis is blocked by p18 and additional regulatory mechanisms or factors which assist in protection of nucleated host cells from MAC attack. Susceptibility to homologous C of these cell lines, therefore, reflects relatively low potency of C regulation on their membrane.
Differential in situ expansion and gene expression of CD4+ and CD8+ tumor‐infiltrating lymphocytes following adoptive immunotherapy in a murine tumor model system
Authors: Evans Robert , Duffy Theodore M. , Kamdar Sonya J.
Published: March 2007

In previous reports, we demonstrated that adoptively transferred T cells homed to the tumor site (among other sites) and that amplification of immune responses occurred in situ leading to the generation of cytotoxic CD8+ tumor‐infiltrating lymphocytes (TIL) and macrophages. The present report extends these findings and shows that following adoptive immunotherapy (AIT) of mice bearing the immunogenic transplanted methylcholanthrene‐induced rhabdomyosarcoma (MCA/76‐9) there was a differential expansion of CD4+ and CD8+ TIL, the numbers peaking on days 6 and 8, respectively. At this time, CD8+ TIL accounted for the majority of Thy‐1+ cells. Northern analyses of RNA extracted from positively selected (by panning) Thy‐1+, CD8+ and CD4+ TIL isolated 8 days after AIT indicated the following: in five separate experiments, CD4+ cells expressed three‐ to sixfold more interleukin (IL)2 mRNA and six‐ to eightfold more IL6 mRNA than CD8+ cells, while CD8+ TIL expressed three‐ to sixfold more IL 2 receptor (IL 2R) mRNA and four‐to sixfold more interferon‐γ mRNA than CD4+ cells. TIL cultured in 10% fetal bovine serum failed to release IL2 over a 24‐h period, whereas both IL6 and interferon‐γ activities were demonstrable. The level of IL2R mRNA expression was reflected by a vigorous proliferative response of CD8+ TIL to exogenous recombinant IL2 and only a low response by CD4+ cells suggesting that most of the CD4+ TIL were in the resting stage. This was confirmed when it was shown that the incubation of panned CD4+ TIL with IL2 supplemented with irradiated spleen cells and “spent” 76‐9 tumor culture supernatant (as a source of antigen) induced expansion of TIL resulting in a population consisting of >90% CD4+ TIL. The overall data suggest that the relatively deactivated state of the CD4+ TIL at this particular time reflects the status of the rejection process in terms of the absence or low concentration of stimulating tumor‐associated antigen.
Polymorphisms and haplotypes in the human immunoglobulin x locus
Authors: Pargent Walter , Schäble Karlheinz F. , Zachau Hans G.
Published: March 2007

By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the x locus (p for the Cx proximal, d for the distal copy). Some of these duplication‐differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B‐Jx‐Cx region were included into the study. Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11. The latter haplotype in which the whole distal copy of the x locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11 are consistent with Mendelian inheritance. Haplotypes N and G are distinguished from each other by eight RFLP markers. Six additional haplotypes, which were found in one or several individuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.
The sgp‐60 mlecule is linked to the plasma membrane via a glycosylphosphatidylinositol anchor
Authors: Cabrero Jesús González , Reiser Hans
Published: March 2007

We have recently identified a novel murine glycoprotein termed sgp‐60, which is expressed on the cell surface of T and B lymphocytes. Because of the profound modulatory effects of sgp‐60 on activation through the T cell receptor/CD3 complex, we have examined the membrane attachment domain of the molecule. sgp‐60 is not expressed on the surface of variants of a T‐T hybridoma cell line that are defective in glycosylphosphatidylinositol (GPI) anchor biosynthesis. In wild‐type but not in mutant cells, sgp‐60 can be labeled with palmitic acid. Furthermore, the molecule can be removed from the cell surface of both T and B lymphocytes by enzymatic digestion with a phosphatidylinositol‐specific phospholipase C. We conclude that the sgp‐60 molecule is linked to the plasma membrane via a GPI anchor.
Characterization of specific T helper cell activity in mice bearing alloantigenic tumors in the anterior chamber of the eye
Authors: Bando Yasuharu , Ksander Bruce R. , Streilein J. Wayne
Published: March 2007

P815 tumor cells injected in the anterior chamber (AC) of eyes of BALB/c mice elicit anterior chamber‐associated immune deviation (ACAID) whereby delayed‐type hypersensitivity (DTH) responses to the tumor‐associated antigens are suppressed, precursors of cytotoxic T cells are clonally expanded but not Triminally differentiated, and levels of tumor‐specific serum antibodies are elevated. These results imply the presence of unique helper T(Th) cell functions in these animals. To identify and describe these cells, we first determined the presence of antigen‐activated lymphocytes in AC tumor‐bearing mice, as well as mice that received tumor cells subconjunctivally (SC), as measured by prolifer‐ative responses of lymphocytes from draining lymph nodes and spleens. In addition, we examined the lymphokine secretion profiles [interleukin (IL) 2, IL4] of antigen‐responsive lymph node and spleen cells in limiting dilution analysis. We found that lymphoid organs of mice primed by the SC route contained high frequencies of antigen‐reactive CD4+ cells that secreted IL2 only, or IL2 plus IL4. In addition, IL2‐secreting CD8+ cells were found. Alternatively, the lymphoid organs of mice receiving AC inoculations of P815 cells contained CD4+ as well as CD8+ cells that secreted IL2 after antigen stimulation. However, no IL4‐secreting cells were found. According to a recent model of differentiation of CD4+ T cells, precursors of Th cells (that secrete IL2 alone) differentiate into Th0 cells that can secrete IL2, IL4 and IFN‐γ. These cells further differentiate into Th1 cells and Th2 cells that secrete IL2 or IL4, respectively. We interpret the absence of IL4‐secreting CD4+ cells in AC tumor bearing mice to mean that in these mice precursor Th cells are unable/prevented from differentiating into Th0 cells.
Regulation of interleukin 2 production, interleukin 2 mRNA expression and intracellular glutathione levels in ex vivo derived T lymphocytes by lactate
Authors: Roth Steffen , Droge Wulf
Published: March 2007

The concentration of L‐lactate in the blood plasma of higher vertebrates is about 1 mM but can be as high as 30 mM under certain physiological and pathological conditions or in the vicinity of glycolytically active cells including macrophages. Here we report that high but physiologically relevant concentrations of lactate increase the expression of interleukin 2 (IL 2)‐specific mRNA and the production of IL2 activity in cultures of mitogenically stimulated T cells. Lactate supports IL 2 production most effectively if added 0–8 h after T cell stimulation and only in cultures of CD4+ but not of CD8+ T cells. In contrast to the DNA synthesis activity in these cell cultures, IL 2 production is not augmented but rather inhibited by glutathione (GSH). Lactate causes a reduction of intracellular GSH levels, and lactate‐containing cultures require accordingly higher extracellular cysteine concentrations than control cultures to achieve similar intracellular GSH levels. In view of the strong variations of extracellular lactate concentrations In vivo, our experiments suggest that lactate may be part of a previously unknown mechanism by which the metabolic microenvironment modulates gene expression in T cells.
Apoptosis is associated with the extensive B cell death in the sheep ileal Peyer's patch and the chicken bursa of Fabricius: A possible role in B cell selection
Authors: Motyka Bruce , Reynolds John D.
Published: March 2007

The ileal Peyer's patch (PP) and the bursa of Fabricius have major roles in populating the B cell system in sheep and chickens, respectively. These tissues contain > 90% B cells and possess a massive proliferation index with > 5% of B cells entering mitosis per hour. Paradoxically, almost all of the B cells produced in these sites rapidly die in situ. Here we show that the extensive B cell death occurring in the ileal PP and bursa is associated with apoptosis. Gel electrophoresis of ileal PP cell DNA from 7–14 week‐old lambs and bursal cell DNA from 4‐week‐old chickens demonstrated a laddering of DNA in multiples of approximately 200 bp, a pattern indicative of apoptosis. In sheep, the intensity of the laddering pattern seen after agarose gel electrophoresis was always greater with ileal PP cell DNA compared with thymocyte DNA, and usually greater than jejunal PP cell DNA. Likewise, DNA isolated from chicken bursal cells and mouse PP cells always exhibited a more intense laddering pattern than chicken or mouse thymocytes, respectively. When placed in culture ileal PP cells died rapidly < 40% viable cells were recovered after 24 h. Within 6 h of culture many ileal PP cells exhibited an apoptotic appearance in that they contained condensed chromatin and fragmented nuclei. Moreover, < 55% of total cellular DNA was fragmented. Compared with thymocytes, ileal PP cells underwent DNA fragmentation to a much greater extent and with a faster time course in short‐term culture. We propose that cell death by apoptosis may make an important contribution to B cell development in the lamb ileal PP and the chicken bursa. Apoptosis may provide a mechanism for the diversification of the B cell immune repertoire and/or the selection of non‐self reactive B cells.
The tyrosine kinase activity of p56lck is increased in human T cells activated via CD2
Authors: Danielian Silvia , Fagard Remi , Alcover Andrés , Acuto Oreste , Fischer Siegmund
Published: March 2007

An early biochemical event associated with T cell activation is tyrosine phosphorylation. We have previously shown that p56lck, a lymphocyte‐specific protein tyrosine kinase, is hyperphosphorylated on serine and tyrosine residues 15 minutes after activation via CD2 with a concomitant shift to a higher molecular mass. We now demonstrate that the tyrosine kinase activity of p56lck is increased within seconds following CD2 triggering. This activity decreases thereafter correlating with the appearance of changes in phosphorylation previously described. These results suggest that p56lck may play an important role in the CD2 activation pathway.
Evidence for idiotypic sharing between conventional and naturally activated B cells
Authors: Song Charles H. , Calandra Gertrudis B. , Miller Alexander , Sercarz Eli E. , Keller Margaret A.
Published: March 2007

In the primary immune response to hen egg‐white lysozyme (HEL), approximately 50% of the primary anti‐HEL antibody‐forming cells (AFC) express IdXE, an idiotype absent from the secondary response. Spontaneous IgM enzyme‐linked immunosorbent assay (ELISA)‐AFC in spleen cells from naive A/J mice were analyzed for the occurrence of IdXE by use of two affinity‐purified rabbit antisera, R213 and R8, each raised against a different primary IdXE+, anti‐HEL mAb. Two ELISA‐AFC assay methods were used: direct coating of immunoplates with R213 or development of IgM‐producing ELISA‐AFC with biotinylated R8. From ages 7 days until 6 months, 10–20% of spontaneous IgM AFC were found to be IdXE+. IdXE+ anti‐HEL IgG1 monoclonal antibodies, 2F4 or 3C11 (100 μg/ml), completely inhibited binding of biotinylated R8 (0.5 μg/ml) to spontaneous IgM‐AFC while IdXE−, anti‐HEL IgG1, 5E11 and 2C7, showed no significant inhibition. Greater than 90% of IdXE+ spontaneous IgM‐AFC were not HEL specific. We conclude that a dominant set of idiotopes found in a conventional antigen‐driven immune response can also play a major role in the spontaneously activated B cell repertoire. Our data argue against a bifurcation of the immune system into a compartment of idiotypic network‐related cells and an independent set of non‐network cells subject to antigenic stimulation.
A quantitative assay of peptide‐dependent class I assembly
Authors: Elvin John , Cerundolo Vincenzo , Elliott Timothy , Townsend Alain
Published: March 2007

We have developed a quantitive assay for the measurement of class I assembly induced by peptide. We have applied this assay to H‐2Db, Kb and HLA‐A2.1 with a panel of 49 overlapping peptides derived from HIV‐1 gag protein. We find that the effects of peptide on assembly form a continuous distribution. By defining positives as those that increase the concentration of folded heavy chains more than three standard deviations from the control we show that 7/48 bind A2.1, 11/49 bind Db and 7/47 bind Kb. The assembly assay contrasts with solid‐phase assays in being more discriminating (fewer peptides binding any given class I molecule), and showing less overlap in the patterns of peptides bound by the three class I molecules.
CD3+ T cells in severe combined immunodeficiency (scid) mice. IV. Graft‐vs. ‐host resistance of H‐2d scid mice to intravenous injection of allogeneic H‐2b (C57BL/6) spleen cells
Authors: Claesson Mogens H. , Rudolphi Angelika , Tscherning Thomas , Reimann Jörg
Published: March 2007

Intraperitoneal injection of 2 × 107 nonfractionated spleen cells (SC) from C57BL/6 (B6, H‐2b) mice into completely allogeneic immunodeficient H‐2d scid mice induced clinical and histological signs of acute graft‐vs.‐host disease (GVHD), with all transplanted severe combined immunodeficiency (scid) mice dying in the 3rd week post‐transfer. In contrast four out of five scid mice survived for > 7 weeks after intravenous (i.v.) injections of equal numbers of B6 SC. Intravenously allotransplanted scid mice analyzed in the 8th week post‐transfer had engrafted donor‐type CD4+ and CD8+ T cells in the spleens but showed no clinical or histological evidence of GVHD. i.v. injection of 107 or 106O B6 SC engrafted allogeneic T cells in spleens of scid recipients; in contrast, i.v. injection of 105 nonfractionated B6 SC or 3 × 105 cell sorter‐purified, naive or anti‐H‐2d‐primed splenic CD4+ or CD8+ B6 T cells led to rejection by young scid recipient mice. B6 T cells engrafted into spleens of scid mice after i.v. injection showed proliferative anti‐host alloreactivity in vitro. No cytotoxic reactivity against host‐type alloantigens was found in standard 4‐h 51Cr‐release assays. These data demonstrate that allogeneic T cells injected i.v. into immunodeficient scid mice are partially tolerized against host‐type alloantigens.
Homotypic aggregation of human cell lines by HLA class II‐, class Ia‐ and HLA‐G‐specific monoclonal antibodies
Authors: Ødum Niels , Ledbetter Jeffrey A. , Martin Paul , Geraghty Daniel , Tsu Theta , Hansen John A. , Gladstone Paul
Published: March 2007

Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well‐established role of class II antigens in low‐affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce adhesion between T and B cells by activating the CD18/CD11a (LFA‐1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA‐DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti‐DQ (TU22, Leu‐10) mAb, induced homotypic aggregation of human class II‐positive monocytic (1937) and T leukemic (HUT78) tumor cell lines and Epstein‐Barr virus (EBV) transformed B‐lymphoid cell lines (EBV‐LCL). Class II‐negative cell lines (U‐937 and the EBV‐LCL mutant line 616) were not induced to aggregate. An HLA‐G‐transfected EBV‐LCL, 221‐AGN, but not the class I‐negative parental line, 221, showed homotypic aggregation in response to an HLA‐G specific mAb (87G) and a broad reacting class I‐specific mAb (IOT2). Both cell lines responded with aggregation to anti‐class II mAb (TU35). The anti‐class I mAb, W6/32, had no effect on all cell lines tested and two anti‐β2‐microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature‐sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase inhibitors (genistein, herbimycin A). Serine/threonine protein kinase inhibitors (staurosporin, H7) partly inhibited the aggregation responses. There was no strict correlation between induction of aggregation and epitope density. FcR were not involved in the aggregation response, since F(ab')2 fragments of anti‐DR mAb, L243, were as effective as the whole antibody. The aggregation was not influenced by mAb against accessory molecules previously shown to be involved directly or indirectly in homotypic aggregation [CD11a (LFA‐1)/CD18/CD54 (ICAM‐1), CD58 (LFA‐3)/CD2, BB1/CD28, CD43, and CD44]. In conclusion, these data provide further evidence that HLA molecules are implicated in a novel, cellular aggregation phenomenon involving the cytoskeleton.
Synovial fluid‐derived Yersinia‐reactive T cells responding to human 65‐kDa heat‐shock protein and heat‐stressed antigen‐presenting cells
Authors: Hermann Elisabeth , Lohse Ansgar W. , Van Der Zee Ruurd , Van Eden Willem , Mayet Werner J. , Probst Peter , Poralla Thomas , zum Büschenfelde Karl‐Hermann Meyer , Fleischer Bernhard
Published: March 2007

Humoral and cellular immune reactions to heat‐shock proteins have been implicated in the pathogenesis of arthritis. Heat‐shock proteins occur in bacteria as well as all eukaryotes and have been highly conserved during evolution. Cross‐reactivity between bacterial and human heat‐shock proteins induced at the site of inflammation may underlie the pathogenesis of some forms of arthritis. In order to test this hypothesis, we raised and cloned a Yersinia‐specific T cell line from the synovial fluid lymphocytes of a patient with Yesinia‐induced reactive arthritis. From this line we obtained a CD4+ T cell clone that proliferated in response to Yersinia antigens and both to the mycobacterial and the human 65‐kDa heat‐shock protein. This T cell clone also proliferated in response to autologous heat‐stressed antigen‐presenting cells as well as to synovial fluid mononuclear cells from the inflamed joint, thus showing true autoreactivity against endogenously synthetized self‐antigen. These results demonstrate the induction of an autoimmune T cell response by a natural bacterial infection and support the important role of heat‐shock proteins in the pathogenesis of immune‐mediated arthritis.
In the mouse the maturation stage of the peripheral CD4+ CD45RA+ subset is different from that of the CD8+ CD45RA+ subset
Authors: Lightstone Elizabeth B. , Wyllie David , Marvel Jacqueline
Published: March 2007

In vitro studies have suggested that the presence of CD45RA on subsets of CD4 and CD8 cells defines naive T cells and that, in response to antigen, CD45RA+ cells become CD45RA− along a differentiation pathway. To test the hypothesis that CD45RA+ cells are naive cells which have just left the thymus, young mice were thymectomized. This would be predicted to lead to a fall in the size of the peripheral pool of CD45RA+ T cells. However, the changes in the size of this pool would also be dependent on the life‐span and self renewal capacity of the CD45RA+ T cells in the periphery. Therefore, to test the contribution of the thymus to the peripheral CD45RA+ pool, the percentage of CD45RA+ cells among spleen lymphocyte subsets was studied from 10 days up to 2 years of age in thymectomized and control mice. We also studied the expression of the memory marker CD44 on the CD45RA subsets of CD4 and CD8 cells, as well as the effect of in vitro activation on expression of CD45RA. Our results show that CD8+ CD45RA+ cells are mainly CD44− and their maintenance is dependent on the presence of the thymus. In contrast, the majority of CD4+CD45RA+ are CD44+ and are not affected by thymectomy. This indicates that the maturation stage of CD8+CD45RA+ cells is different from that of CD4+CD45RA+ cells.
Dissociation between plasma and monocyte‐associated cytokines during sepsis
Authors: Munoz Carlos , Misset Benoit , Fitting Catherine , Blériot Jean‐Pierre , Jean‐Carlet  , Cavaillon Jean‐Marc
Published: March 2007

We report our investigations of circulating interleukin (IL) 1β, IL 6 and tumor necrosis factor (TNF)‐α, as well as cell‐associated IL 1α, IL 1β and TNF‐α in plasma and monocytes of 21 patients with sepsis syndrome and 6 patients with non‐septic shock. Longitudinal studies reveal that (a) the most frequent detectable plasma cytokines were TNF‐α and IL 6, (b) the presence and the kinetics of circulating cytokines were independent of one other, (c) detectable levels of cytokines could be found for a long period of time, and (d) significantly higher levels of IL 6 were found for non‐surviving patients. Because of the in vivo half‐life of cytokines and of the existence of numerous specific high‐affinity receptors, it is quite probable that detectable plasma cytokines represent the excess of produced mediators which have not been trapped by the target cells. TNF‐α (410 ± 65 pg/106 monocytes) and IL 1β (153 ± 60 pg/106 monocytes) were frequently found associated to monocyte lysates (88% and 50%, respectively). Despite the fact that IL 1α is the most abundant cytokine found associated to monocytes following in vitro activation, IL 1α was rarely found in monocytes of intensive care unit patients (29%). No correlation was found to exist between the levels of plasma cytokines and cell‐associated cytokines. Some patients had plasma TNF‐α or IL 1β in the absence of the corresponding monocyte‐associated cytokine. This observation suggests that cells other than monocytes can participate in the production of circulating cytokines. At the end of the longitudinal study (day 14 ± 2), only 2/12 surviving patients still had plasma TNF‐α, whereas 8/12 had monocyte‐associated TNF‐α. These results indicate that activation of monocytes still occurs in patients for whom no plasma cytokines can be detected. Thus, in addition to the measurement of plasma cytokine, measurement of cell‐associated cytokine appears useful to assess cytokine production and monocyte activation in vivo.