bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal CD45RC expression and elevated CD45 protein tyrosine phosphatase activity in LEC rat peripheral CD4+ T cells
Authors: Sakai Tohru , Agui Takashi , Matsumoto Kozo
Published: March 2007

LEG rats are known to show a maturational arrest in the development of CD4+8+ to CD4+8− cells in the thymus. Despite the blockade of maturation of CD4+8− thymocytes, CD4+ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin‐2 (IL‐2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4+ cells in normal rat highly expressed CD45RC (CD45RChigh), the level of CD45RC expression was low (CD45RClow) in LEC rat peripheral CD4+ cells. However, CD4+ cells from both strains highly expressed CD45 when those cells were stained by pan‐CD45 mAb, suggesting that LEC rat CD4+ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 × LEC)F1 × LEC were examined, the phenotype for CD45 expression pattern in CD4+ cells was clearly correlated with IL‐2 production level in response to Con A stimulation. Thus, CD45RClow cells exhibit a defect in IL‐2 production, while CD45RChigh cells show normal IL‐2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4+ cells was threefold higher than that of normal rat CD4+ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100‐ and 40‐kDa proteins, in normal rat CD4+ cells. In LEC rat CD4+ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL‐2 production in LEC rat peripheral CD4+ T cells.
Both membrane‐bound and soluble forms of CD14 bind to Gram‐negative bacteria
Authors: Jack Robert S. , Grunwald Uwe , Stelter Felix , Workalemahu Grefachew , Schütt Christine
Published: March 2007

Tissue macrophages and their precursors – the blood monocytes – respond rapidly to a bacterial infection with the release of inflammatory mediators. These mediators are involved in the recruitment of phagocytic cells, principally neutrophils, from the blood to the site of infection. To initiate this process macrophages and monocytes must be able to detect the presence of bacteria in a reliable, but nevertheless nonspecific, fashion. It is thought that this is achieved by means of receptors on the cell surface which recognize structures common to many different bacteria. One candidate for such a “pattern recognition element” is the cell surface glycoprotein CD 14. CD 14 has been shown to bind components of the Gram‐positive cell wall and it also binds soluble lipopolysaccharide released from Gram‐negative bacteria. In both cases the interaction with CD 14 leads to an activation of the cell. Here we show that human peripheral blood monocytes can, in addition, bind intact Gram‐negative bacteria in the presence of serum and that this process involves CD14. When CD14 expression is induced on the myelomonocytic cell line U937 by treatment with vitamin D3 the cells concomittently acquire the capacity to bind bacteria. Furthermore, a non‐monocytic cell line which does not bind bacteria acquires the capacity to do so when transfected with either the human or mouse CD14 gene. This binding can be inhibited by blocking the CD14 receptor with anti‐CD14 antibody or by blocking the ligand on the bacteria with soluble CD14. Finally we demonstrate binding of sCD14 to Escherichia coli. We conclude that in the presence of serum both membrane‐bound and soluble forms of CD14 can bind to Gram‐negative bacteria. This suggests that CD14 may play a role in the detection and elimination of intact bacteria in vivo.
Impaired survival of T cell receptor Vγ3+ cells in interleukin‐4 transgenic mice
Authors: Erb Klaus Joseph , Hanke Thomas , Schimpl Anneliese , Hünig Thomas , Stingi Georg , Elbe Adelheid
Published: March 2007

The mouse epidermis contains a network of Thy‐1+ dendritic T cells. Most of these cells express a homogeneous T cell receptor (TCR) configuration (Vγ3/ Vδ1) with only negligible junctional diversity. Because fetal thymocytes are precursors of these dendritic epidermal T cells (DETC) and the addition of interleukin (IL)‐4 to fetal thymic organ cultures causes an early arrest in thymopoiesis, we examined DETC development in transgenic (tg) mice expressing IL‐4 under the control of major histocompatibility complex class I regulatory sequences. Immunohistologic examination of epidermal sheets and polymerase chain reaction analysis of total skin RNA from IL‐4 tg mice failed to reveal TCR Vγ3+ DETC and Vγ3 mRNA, respectively. In contrast, the sizes of TCR γδ subpopulations in lymphoid organs were unchanged in these mice. Although the numbers and staining intensities of TCR Vγ3+ thymocytes in early fetal (days 14–17) IL‐4 tg mice were similar to those of littermate controls, we observed a preferential death of these cells in thymic organ cultures from IL‐4 tg mice. We observed further that epidermal sheets prepared from 9‐day‐old mice whose mothers had been treated with an IL‐4‐neutralizing antibody from day 12 to day 18 of pregnancy contained DETC numbers similar to those of controls. However, upon termination of the anti‐IL‐4 treatment, DETC ceased to expand. We conclude that IL‐4 impairs the survival of TCR Vγ3+ cells.
Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen: involvement of marginal metallophilic macrophages in the uptake of immune‐stimulating complexes
Authors: Claassen Ivo J. T. M. , Osterhaus Ab D. M. E. , Claassen Eric
Published: March 2007

Several mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune‐stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system. However, until now no method for the subcellular detection of iscom in situ was available. In the present study, a novel, fast and simple method for the detection of iscoms in situ is demonstrated. By making use of the lipophilic fluorescent carbocyanine dyes, 1,1‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (Dil) and 3,3′‐dioctadecyloxacarbocyanine perchlorate (DiO), rabies virus antigen and iscom prepared with this antigen were visualized with fluorescence microscopy. The labeled antigen and iscoms were observed in macrophages of spleen and liver of mice within 1–2 h after intravenous administration. When administered intramuscularly or in the footpad, uptake in macrophages of draining lymph nodes could be demonstrated. In the spleen, labeled inactivated virus antigen localized preferentially in the marginal zone macrophages and to a lesser extent in the red pulp macrophages. In contrast, antigen presented in iscom was taken up mainly by the marginal metallophilic macrophages and to a much lesser extend by marginal zone macrophages or follicular‐dendritic and ‐B cells. This method enables the detection of iscom and membrane viruses and allows the analysis of their relation to antigen‐presenting cells in situ. Here, we demonstrate that iscom containing rabies virus antigen are taken up by a subset of macrophages in the spleen distinct from those that take up inactivated rabies virus antigen not presented in iscom, thereby possibly explaining the observed difference in immunogenicity of these antigen preparations. Furthermore, we show a lower efficiency on the induction of humoral and cellular responses after intravenous immunization for both types of antigen when compared with subcutaneous immunization.
Mapping and ranking of potential cytotoxic T epitopes in the p53 protein: effect of mutations and polymorphism on peptide binding to purified and refolded HLA molecules
Authors: Gnjatic Sacha , Paillerets Brigitte Bressac‐De , Guillet Jean‐Gérard , Choppin Jeannine
Published: March 2007

In many cancer cells, the p53 gene displays point mutations that result in stabilization and accumulation of the p53 protein. Therefore, p53 peptides could be presented to the immune system by tumor cells; thus, p53 might be a suitable target antigen for developing an immunotherapy against tumors using cytotoxic T lymphocytes (CTL). To map candidate CTL epitopes, we synthesized 150 peptides of 8–11 residues that contained putative anchor motifs required for binding to common HLA class I molecules. They were tested for their capacity to promote the assembly of purified and refolded HLA‐A1, A2, B7 and B8 molecules. The following wild‐type p53 peptides were found to be reactive with the HLA molecules tested: 196–205 and 226–234 bound moderately to HLA‐A1; 25–35, 65–73, 129–137, 187–197, 263–272 and 264–272 bound strongly, and 187–195 and 256–264 moderately to HLA‐A2; 26–35, 63–73, 189–197, 249–257 and 321–330 bound strongly to HLA‐B7; and 135–143, 210–218 and 375–383 bound weakly to HLA‐B8. We also analyzed the effects of p53 mutations occurring naturally in tumors on peptide/HLA assembly. We found substitutions that enhanced, diminished or had no effect on the peptide binding to HLA molecules. Polymorphism at position 72 mainly affected peptide/HLA‐B7 binding, the proline allele P72 giving a less‐reactive peptide (63–73) than the arginine allele R72. We have ranked potential p53 epitopes according to their reactivity for purified HLA molecules, allowing the selection of appropriate peptides and HLA molecules to attempt CTL induction in vitro.
Heterogeneity of rat encephalitogenic T cells elicited by variants of the myelin basic protein (68–86) peptide
Authors: Sun Deming , Coleclough Christopher , Hu Xian‐Zhen
Published: March 2007

By immunizing Lewis rats with myelin basic protein (MBP) peptide variants derived from the major encephalitogenic epitope of guinea pig (MBP(68–88) and then isolating encephalitogenic T cells from these animals, we demonstrated that the variant peptides do not elicit the same encephalitogenic T cell subsets as those induced by the wild‐type peptide or by intact MBP. Rather, the pathogenic T cells differed in clonal composition as reflected by their heterogeneous responses to a panel of variant peptides and by their T cell receptor usage. Thus, molecules mimicking the MBP(68–88) autoantigen can elicit pathogenic T cell subsets without necessarily cross‐reacting with T cells specific for the original autoantigen. This suggests that a more clonally diverse group of pathogenic T cells might be involved in EAE than has been apparent from studies with intact MBP or its unaltered peptides.
Involvement of cAMP in CD3 T cell receptor complex‐ and CD2‐mediated apoptosis of human thymocytes
Authors: Mentz Frank , Mossalayi M. Djavad , Ouaaz Fateh , Debré Patrice
Published: March 2007

During intrathymic T cell development, elimination of autoreactive T cell clones by programmed cell death (PCD or apoptosis) is an essential mechanism for self tolerance. The precise intracellular second messengers that lead to this process remain to be determined. In the present work, we show that treatment of freshly isolated thymocytes with an antagonist of the cAMP pathway, the Rp‐cAMP, significantly decreases spontaneous death by apoptosis of human thymocytes in vitro. Addition of Rp‐cAMP also rescues thymocytes from activation‐induced apoptosis following the ligation of surface CD3/T cell receptor complex or CD2 antigens. A cAMP analog, the dibutyryl(Dibut)‐cAMP increases PCD of human thymocytes in a dose‐dependent manner. Growth and rescue from PCD of thymocytes in the presence of interleukin (IL)‐2 or IL‐4 are also enhanced by Rp‐cAMP and inhibited by Dibut‐cAMP. Finally, we detect substantial levels of intracellular cAMP in freshly isolated thymocytes. This study reveals the involvement of cAMP as a second messenger during the apoptosis of normal human thymocytes.
The influence of invariant chain on the positive selection of single T cell receptor specificities
Authors: Tourne Sylvie , Nakano Naoko , Viville Stéphane , Benoist Christophe , Mathis Diane
Published: March 2007

The appearance of peptide‐loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4+ T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain‐deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single‐positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of Ek. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti‐MCC 88‐103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene‐encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptide in vitro. The most likely interpretation of these findings is that selection of most CD4+ T cells depends on MHC class II complexes loaded with peptide in an Ii‐dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii‐independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4+ T cells, for which there exists little prior evidence.
Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ52‐JH region of the immunoglobulin heavy chain locus
Authors: Taki Shinsuke , Schwenk Frieder , Rajewsky Klaus
Published: March 2007

We investigated gene rearrangements in the mutant IgH locus of a mouse strain generated by insertion of a rearranged heavy chain variable region gene (VT15) into the DQ52‐JH region through gene targeting. In more than half of the B cells of heterozygous mutant mice, the mutant IgH locus was silenced by the rearrangement of an endogenous DH or DH and VH gene to the inserted VT15 gene. In these cases, a functional VHDHJH gene was present on the wild‐type allele. The silencing rearrangement appeared to be mediated by recombination signal sequence (RSS)‐like elements present in the “recipient” VT15 gene. Among the many such elements on the inserted VT15 gene, which apparently met the requirement for an RSS with respect to nucleotide sequence, only two were observed in the actual rearrangements. This indicates that targeting of the recombination machinery involves sequences in addition to the RSS motifs as they have been characterized so far. In homozygous mutant mice, most B cells appeared to carry the intact VT15 gene on both mutant IgH alleles, although single‐cell polymerase chain reaction revealed that silencing rearrangements occured frequently in B cell progenitors in the bone marrow. This observation indicates that once silencing rearrangements are initiated in a cell, they involve both VT15 genes in most cases, reminiscent of normal DH‐JH rearrangement. B cells which did not initiate such rearrangements develop to populate the peripheral B cell compartment.
Sphingomyelin‐ceramide turnover in CD28 costimulatory signaling
Authors: Chan Gordon , Ochi Atsuo
Published: March 2007

When the CD28 membrane glycoprotein of T cells binds to its ligand, a signal is transmitted that is required for T cell receptor‐induced proliferation and cytokine secretion; T cells are not stimulated by the CD28 signal alone. Ligation of CD28 initiated sphingomyelin hydrolysis and generated ceramide. Treatment of T cells with either exogenous sphingomyelinase or a cell‐permeable ceramide analogue, C6‐ceramide, mimicked the CD28 signal by inducing T cell proliferation and interleukin‐2 gene transcription. Stabilization of interleukin‐2 mRNA was also observed in C6‐ceramide‐treated cells. Thus, the sphingomyelin‐ceramide pathway is a candidate for mediating the costimulatory signal.
Association of the transmembrane 4 superfamily molecule CD53 with a tyrosine phosphatase activity
Authors: Carmo Alexandre M. , Wright Mark D.
Published: March 2007

Cell surface proteins of the transmembrane 4 superfamily (TM4SF) are a newly characterized family of proteins which are presumed to span the plasma membrane four times. The function of this family of molecules is poorly understood, but based on monoclonal antibody studies there is some evidence that they may be involved in transmembrane signal transduction and regulation of cell proliferation, differentiation, or both, in a number of different cell types. CD53 is a member of this family that is expressed on leukocytes, and transduces activation signals through unknown mechanisms that may involve phosphorylation events. However, CD53 has never been shown to associate directly with kinases. Here, we show by immunoprecipitation from cell lysates of lymph nodes and a thymoma cell line, that immune complexes of rat CD53 contain tyrosine phosphatase activity. The CD53‐associated phosphatase was able to dephosphorylate in vitro the phosphorylated tyrosine kinase Lck, as well as a synthetic substrate, and its activity was abrogated by a tyrosine phosphatase inhibitor. Although its identity has not been established, it is clear from depletion experiments that it is not CD45. CD63, a second member of the TM4SF, also co‐precipitates a phosphatase activity from rat basophilic leukemia cells. These results demonstrate that the TM4SF members associate with tyrosine phosphatases. It seems possible that such associated phosphatases may contribute to the signal transduction capacity of TM4SF molecules.
Differential contribution of the FcRγ chain to the surface expression of the T cell receptor among T cells localized in epithelia: analysis of FcRγ‐deficient mice
Authors: Park Seung Yong , Arase Hisashi , Wakizaka Keisuke , Hirayama Nakami , Masaki Shigehiro , Sato So‐Ichiro , Ravetch Jeffrey V. , Saito Takashi
Published: March 2007

The function of the Fc receptors γ chain (FcRγ) for the expression of the T cell receptor (TCR) complex and for T cell development, especially for T cells localized in epithelia, was investigated by analyzing FcRγ‐deficient mice. In wildtype mice, CD8αα+β−TCRαβ+ T cells of intestinal intraepithelial lymphocytes (i‐IEL) utilized CD3ζ homodimers and ζ‐FcRγ heterodimers, whereas CD8α α+β−TCRγδ+ i‐IEL used ζ‐FcRγ and FcRγ homodimers in the TCR complex. On the other hand, these T cells in FcRγ‐deficient mice contained only ζ homodimers. The surface expression of the TCR complex was reduced in CD8αα+β−i‐IEL and dendritic epidermal T cells (DETC) in these mice, whereas the development of these T cells was normal. The degree of reduction appeared to depend on the expression level of FcRγ. In contrast to these populations, TCRγδ+ intraepithelial T cells in reproductive organs (r‐IEL) were dramatically decreased, suggesting that the development of r‐IEL is FcRγ‐dependent, probably due to the predominant usage of FcRγ homodimers in the TCR complex. These results indicate that the FcRγ chain contributes differently to the TCR expression and to the development of T cells localized in epithelia.
Differences in processing of an autoantigen by DR4:Dw4.2 and DR4:Dw14.2 antigen‐presenting cells
Authors: Nicolle Michael W. , Hawke Simon , Willcox Nicholas , Vincent Angela
Published: March 2007

Variations in antigen processing can influence class II‐restricted T cell responses. We now report a highly significant difference (p < 0.001) between the ability of antigen‐presenting cells from three HLA‐DR4:Dw14.2 (Arg71) and six DR4:Dw4.2 (Lys71) individals to present recombinant or native acetylcholine receptor antigens to a myasthenia gravis T cell clone. The difference was greatest with longer antigens, and not seen with short synthetic peptides, suggesting that it may result from a difference in antigen processing between the two alleles. The results were not related to the presence of myasthenia gravis or of steroid therapy. They could, however, be of relevance in rheumatoid arthritis where particularly severe disease associates with Dw4.2/Dw14.2 heterozygosity.
Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin
Authors: Girolomoni Giampiero , Lutz Manfred B. , Pastore Saveria , Aßmann Caroline U. , Cavani Andrea , Ricciardi‐Castagnoli Paola
Published: March 2007

During ontogeny, the skin is progressively populated by major histocompatibility complex class II‐negative dendritic cell (DC) precursors that then mature into efficient antigen‐presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR‐mycMH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum‐free medium was promoted by granulocyte/macrophage colony‐stimulating factor (GM‐CSF), but not macrophage‐CSF. FSDC expressed strong surface‐membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H‐2d.b+, I‐Ad.b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER‐MP 12−, Sca‐1+, Sca‐2+, NLDC‐145−, B7.2+, B7.1−, J11d−, B220−, Thy‐1−, and CD3−. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed‐leukocyte reaction, and markedly increased this function after treatment with GM‐CSF, GM‐CSF and interleukin (IL)‐4 or interferon‐γ (IFN‐γ); in contrast, stem cell factor, IL‐1α and tumor necrosis factor‐α had no effect. Preculture with IFN‐γ was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC‐like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.
Functional discrepancies between tumor necrosis factor and lymphotoxin α explained by trimer stability and distinct receptor interactions
Authors: Schuchmann Marcus , Hess Sigrun , Bufler Philip , Brakebusch Cord , Wallach David , Porter Alan , Riethmüller Gert , Engelmann Hartmut
Published: March 2007

Tumor necrosis factor (TNF) and lymphotoxin α (LTα) are closely related cytokines which bind with nearly identical affinities to the same pair of cell surface receptors, p55 and p75TNFR. Therefore it is assumed that TNF and LTα are redundant cytokines. This study, however, demonstrates that TNF and LTα differ significantly with regard to their mitogenic and cytotoxic potentials. LTα's superior mitogenic effect could be explained by its formation of a more stable trimer. In contrast to the TNF trimer, which disintegrated under physiological conditions into biologically inactive monomers, the LTα trimer remained stable for several days. Accordingly, LTα more effectively induced fibroblast growth which demands long‐term presence of the cytokine. TNF's superior cytotoxicity, which requires only short‐term impact of the cytokine, could be attributed to a distinct interaction with the human p55TNFR. This was demonstrated in NIH 3T3 cells transfected with the human p55TNFR, where cytotoxicity is mediated exclusively by the transfected receptor. Although the p55TNFR had virtually identical affinities for TNF and LTα, as defined by Scatchard analysis, it nevertheless discriminated between binding of each cytokine and showed a 200‐fold enhanced cytotoxicity mediated by TNF.
Plasma cell tumors decrease CD23 mRNA expression in vivo in murine splenic B cells
Authors: Ruzek Melanie C. , Mathur Ambika
Published: March 2007

CD23, the low‐affinity Fc receptor for IgE, is constitutively expressed on mature, naive B cells, but is lost following B cell activation. We and others have shown that CD23 expression on B cells decreases in mice bearing plasma cell tumors. In contrast to these findings, we find that IgE‐secreting tumors do not cause a loss of CD23 expression on host splenic B cells. Decreased expression of CD23 on B cells induced by plasma cell tumors requires direct contact between tumor cells and B cells; and other host cells are not involved. The loss of surface CD23 expression is associated with a decrease in steady‐state CD23 mRNA levels in B cells from plasma cell tumor‐bearing mice. Interestingly, loss of CD23 mRNA is observed even in B cells from mice with IgE‐secreting tumors, where we find surface CD23 protein expression to be similar to that of normal mice. The maintenance of surface CD23 expression on B cells in mice with IgE‐secreting tumors is dependent solely on the presence of IgE, and is not a tumorspecific effect. Therefore, we conclude that in vivo, IgE secreted by plasma cell tumors can result in the maintenance of normal levels of surface CD23 expression by a post‐transcriptional mechanism, even when the tumor induces a down‐regulation of CD23 mRNA levels.
Immunosuppression by Fc region‐mismatched anti‐T cell antibody treatment
Authors: Thierfelder Stefan , Mocikat Ralph , Mysliwietz Josef , Lindhofer Horst , Kremmer Elisabeth
Published: March 2007

Formation of anti‐antibodies (anti‐Ab) is known to counteract immunotherapy with anti‐T cell antibodies. Our previously described immunological approach prevented anti‐Ab with the consequence of prolonged survival of fully mismatched skin grafts in C57BL/6 mice. These mice were treated with a single priming injection of a monoclonal anti‐T cell Ab followed by repeated injections of anti‐T cell mAb differing in species origin from the priming mAb. We now show prolonged tolerance to discordant xenogeneic, to bispecific, and even to polyclonal Ab, and demonstrate that the underlying immunosuppressive principle is due to a difference in heavy chain constant region between first and second antibodies, independent of whether or not they share the same idiotype. To examine this phenomenon, a panel of mAb was generated which share the same mouse anti‐Thy‐1.2 idiotype, but carry a human IgG1(T23), IgG3(T212C8), or mouse IgG2a(MmT1) constant heavy chain region. We found that sequential injection of MmT1 and T23 according to the above treatment schedule induced huIgG1 isotype‐specific tolerance to T23, which was similar to that seen when using a primary mAb (MmT5) that was, instead, fully mismatched with T23 in both idiotype and constant region. Thus, differences of idiotype between primary and booster Ab were inconsequential for their ability to inhibit anti‐Ab formation. This novel form of induced specific tolerance to anti‐T cell Ig survived graft rejection and was still evident 230 days after termination of the T cell depletion protocol. Taken together, these results demonstrate that rechallenge with Fc region‐mismatched Ab opens an immunological window that allows for induction of tolerance to immunogenic anti‐T cell Ab and prolonged immunosuppression.
Interleukin‐12 increases interleukin‐4 production by established human ThO and Th2‐like T cell clones
Authors: Jeannin Pascale , Delneste Yves , Life Paul , Gauchat Jean‐François , Kaiserlian Dominique , Bonnefoy Jean‐Yves
Published: March 2007

Interleukin (IL)‐12 is a potent inducer of cell‐mediated immunity: it favors the generation of interferon (IFN)‐γ‐producing T cells, increases IFN‐γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL‐12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL‐12 on the profile of lymphokines produced by established IL‐4‐producing Th0 and Th2‐like human T cell clones (TCC) and by polyclonal T cells. We found that IL‐12 (i) enhances, as previously reported, IFN‐γ production by Th0 TCC and, to a smaller extent, by Th2‐like TCC, (ii) increases the proliferation of Th0 and Th2‐like TCC and (iii) unexpectedly, synergizes with T cell receptor‐associated or nonspecific stimuli in increasing IL‐4 production by these TCC. Thus, IL‐12 potentiates the production of IFN‐γ and also of IL‐4 by established IL‐4‐producing TCC. Although IL‐12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL‐12 may on the contrary potentiate an established Th2 response in humans.
Regulation of cell surface APO‐1/Fas (CD95) ligand expression by metalloproteases
Authors: Mariani Sara M. , Matiba Bernd , Bäumler Caroline , Krammer Peter H.
Published: March 2007

APO‐1/Fas (CD95) ligand (APO‐1L) induces apoptosis in sensitive target cells. Activation‐induced T cell death and Ca2+‐independent cytotoxicity in perforin knockout mice are mediated by APO‐1L. To define whether APO‐1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti‐APO‐1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO‐1L of approximately 42 and 40kDa. In addition, the Ab recognized the non‐glycosylated form of APO‐1L of approximately 32‐33 kDa. In activated human T cells, the soluble form of APO‐1L was detectable with a moleculas mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12‐myristate 13‐acetate/ionomycin induced a significant increase in cell surface APO‐1L only in the presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevented the increase in surface APO‐1L observed in the presence of 1,10‐phenanthroline. Blocking of other classes of proteases (serine‐ and acid‐proteases, chymotrypsin) had no effect. Increased expression of surface APO‐1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO‐1L expression. These results suggest that cell surface expression of human APO‐1L is regulated by Zn2+‐dependent metalloproteases. Cleavage of surface APO‐1L may act as a regulatory mechanism to prevent accumulation of the membrane‐bound form and may cause systemic effects of the APO‐1L.
Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin‐13
Authors: Lebel‐Binay Sophie , Laguerre Brigitte , Quintin‐Colonna Françoise , Conjeaud Hélène , Magazin Marilyn , Miloux Brigitte , Pecceu Florence , Caput Daniel , Ferrara Pascual , Fradelizi Didier
Published: March 2007

Cytokines locally delivered to the site of a tumor boost both specific and nonspecific host anti‐tumor defenses. Interleukin (IL)‐13 is a recently described cytokine produced by mouse type 2 helper T lymphocytes. The aim of this study was to evaluate the inhibition of tumor growth induced by IL‐13 delivered locally within or around transplanted tumor cells in mice. We observed that local administration of IL‐13 at the site of transplanted tumor cells in vivo had potent inhibitory effects on growth of both immunogenic (P815 mastocytoma, H‐2d) or nonimmunogenic (3LL lung carcinoma, H‐2b) tumor cells. Mice injected with transfected P815 cells secreting large amounts of IL‐13 rejected the P815 tumor and developed systemic specific anti‐tumor immunity leading to long‐lasting specific anti‐tumor protection. Less efficient anti‐tumoral effects were obtained with the nonimmunogenic 3LL tumor model when local administration of IL‐13 was achieved by co‐inoculating xenogeneic chinese hamster ovary (CHO) IL‐13 cells. Several local injections of CHO IL‐13 cells were needed to obtain rejection of 3LL tumors and no induction of long‐lasting anti‐3LL memory was obtained. Several studies were performed to elucidate the IL‐13 anti‐tumoral effects. Experiments with nude mice indicated that IL‐13 can also stimulate nonspecific anti‐tumor defenses. The histological examination of P815 IL‐13 cells undergoing rejection showed monocytic cells and neutrophils infiltrating the tumor. Studies indicated that IL‐13 administered in vitro did not directly stimulate the cytotoxicity of peritoneal macrophages and natural killer cells. However, experiments with Boyden chemotaxis chambers indicated that IL‐13 was chemotactic for macrophages. Finally, preliminary experiments in vitro suggest that IL‐13 improved antigenic presentation of P815 membranes. Thus, anti‐tumor effects of IL‐13 in vivo most probably result from pleiotropic effects including recruitment of nonspecific cells and improved stimulation of immune‐specific anti‐tumor effectors.
Follicular dendritic cells and the maintenance of IgE responses
Authors: Helm Shirley L. T. , Burton Gregory F. , Szakal Andras K. , Tew John G.
Published: March 2007

Antigen (Ag) is retained for long periods of time in secondary lymphoid tissues in the form of immune complexes on follicular dendritic cells (FDC). Ag retained on FDC is thought to play a role in maintaining antibody (Ab) responses in vivo. A model for study of Ab production induced by retained Ag in vitro is the spontaneous Ab response. In this response, specific Ab production is induced spontaneously (no exogenous Ag needed) in cultures derived from secondary lymphoid tissues containing persisting Ag. Specific IgG is spontaneously induced and we reasoned that FDC may also play a role in the maintenance of specific IgE responses. To test this hypothesis, we monitored spontaneous antiovalbmin (OVA) IgE production in cultures of lymph node (LN) fragments from OVA‐immunized mice. In addition, highly enriched preparations of OVA bearing FDC were added to OVA‐specific memory cells in an attempt to stimulate OVA‐specific IgE production. Months after secondary immunization, anti‐OVA IgE responses were spontaneously induced when fragments from draining LN were placed into culture. Furthermore, FDC bearing OVA from draining LN induced anti‐OVA IgE production when incubated with spleen cells from OVA‐immune mice whereas identical cultures with FDC bearing environmental Ag from non‐draining LN of the OVA immune animals did not. The anti‐OVA IgE responses were elicited only in cultures containing OVA‐immune memory cells indicating that specific memory cells were critical for these anti‐OVA IgE responses. Removal of FDC from cultures with an FDC‐specific mAb dramatically decreased anti‐OVA IgE production. These studies demonstrate that FDC can induce specific memory T and B cells to produce IgE and help support the concept that FDC‐associated antigen may be involved in the long‐term maintenance of specific IgE responses.
Reciprocal homologous recombination in or near antibody VDJ genes
Authors: Umar Asad , Gearhart Patricia J.
Published: March 2007

To determine if rearranged heavy chain variable (VDJ) genes can recombine with each other by crossing over of DNA strands, we constructed a transgene that contained a promoter, VDJ gene, reporter gene to detect crossover events, intron enhancer, matrix attachment region, and constant gene for IgM (Cμ). Following immunization of transgenic mice, hybrid molecules were isolated from B cell DNA which contained the transgene recombined with the endogenous IgH locus. Reciprocal products of crossovers were detected by plasmid rescue and PCR amplification, and they were sequenced. Recombination occurred somewhere within 147 bp of homology that contained the JH4 gene segment and 3′ flanking DNA. The recombined transgenes had a 20‐fold increase in mutation in the VDJ region compared to nonrecombined transgenes, which indicates that DNA sequences 3′ of the Cμ gene in the endogenous IgH locus are necessary for full activity of the mutator mechanism. The recovery of recombinants between VDJ transgenes and endogenous VDJ genes raises the possibility that reciprocal recombination may somatically diversify rearranged genes between maternal and paternal alleles.
Monoclonal nonspecific suppressor factor β inhibits interleukin‐4 secretion by a type‐2 helper T cell clone
Authors: Nakamura Morihiko , Xavier Ricardo M. , Tanigawa Yoshinori
Published: March 2007

Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine product of a murine T cell hybridoma that inhibits the generation of lipopolysaccharide‐induced immunoglobulin secreting cells in an antigen‐nonspecific manner. Most recently, a cDNA clone encoding MNSFβ, a subunit of MNSF, was isolated and sequenced (Nakamura et al., Proc. Natl. Acad. Sci. USA 1995. 92: 3463). The MNSFβ cDNA encodes a 14.5‐kDa ubiquitin‐like protein showing several MNSF activities. We have previously observed that hybridoma‐derived native MNSF can inhibit interleukin (IL)‐4 secretion by bone marrow‐derived mast cells. Therefore, we studied whether MNSFβ is also able to inhibit IL‐4 secretion by the type‐2 helper T cell clone D10. G4.1. MNSFβ inhibited both the IL‐4 secretion and the proliferation of concanavalin A‐activated D10 cells, although neither effect was complete.
CD40‐mediated lymphotoxin α expression in human B cells is tyrosine kinase dependent
Authors: Worm Margitta , Geha Raif S.
Published: March 2007

The cytokine lymphotoxin (LT)α is known to play a role in B cell activation. As the engagement of the B cell antigen CD40 is known to lead to B cell proliferation and differentiation, we studied LTα expression in human B cells after CD40 ligation. We demonstrate that anti‐CD40 monoclonal antibody (mAb) induces strong LTα mRNA and surface expression in human tonsil B cells. Induction of LTα mRNA and surface expression by CD40 ligation is inhibited by the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein in a dose‐dependent manner. The protein kinase C (PKC)‐specific inhibitors sphingosine and bisindolylmaleimide caused negligible inhibition of anti‐CD40‐induced LTα mRNA and surface expression. No inhibition is observed with the protein kinase (PKA) inhibitors H89 and HA1004. Cross‐linking of the transmembrane phosphatase CD45 to CD40 by using goat‐anti‐mouse F(ab')2 fragments strongly inhibits CD40‐mediated LTα expression in human B cells, confirming the role of PTK activation in CD40‐mediated induction of LTα expression. Inhibitors of the serine/threonine protein phosphatases PP1 and PP2A, okadaic acid and calyculin induce LTα mRNA expression. In contrast, cyclosporin A, an inhibitor of the serine/threonine phosphatase calcineurin has no effect on anti‐CD40‐induced LTα expression. These results suggest that induction of LTα expression in B cells following engagement of CD40 involves activation of protein tyrosine kinases.
Differential effects of interleukin‐10 on the expression of HLA class II and CD1 molecules induced by granulocyte/macrophage colony‐stimulating factor/interleukin‐4
Authors: Thomssen Henrike , Kahan Melvyn , Londei Marco
Published: March 2007

Interleukin (IL)‐10 down‐regulates HLA class II molecules, whether constitutively expressed or up‐regulated by interferon‐γ or IL‐4 on monocytes but not on B lymphocytes. In this study we show that IL‐10 does not inhibit HLA class II expression induced by the combination granulocyte/macrophage colony‐stimulating factor and IL‐4 on monocytes, although it simultaneously abrogates the expression of CD1 molecules induced by the same combination of cytokines. CD1 molecules can act as element of genetic restriction for CD4− CD8− T lymphocytes, and the suppression of CD1 expression by IL‐10 abolished antigen presentation to CD1‐restricted CD4− CD8− T cell receptor‐positive T cells. Although HLA class II expression was not down‐regulated by IL‐10, the antigen specific proliferative response of CD4+ T cells was nevertheless decreased. This was not caused by down‐regulation of known co‐stimulatory molecules such as B7.1, B7.2 and ICAM‐1. IL‐10 decreased the antigen specific proliferative response further by directly influencing the T lymphocytes. Our results indicate that IL‐10 exerts some of its immunoregulatory functions by differential modulation of antigen presenting molecules, induced by the same combination of cytokines.
Peptide‐specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K‐ras p21
Authors: Abrams Scott I. , Dobrzanski Mark J. , Wells Donald T. , Stanziale Stephen F. , Zaremba Sam , Masuelle Laura , Kantor Judith A. , Schlom Jeffrey
Published: March 2007

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host‐derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4+ T helper type 1 (Th1) subset. BALB/c mice (H‐2d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An αβ T cell receptor‐positive, CD3+, CD4+, CD8− T cell line was established, which expressed peptide‐specific proliferation. Cytokine assays revealed the production of interleukin‐2, interferon‐γ, tumor necrosis factor and granulocytemacrophage colony‐stimulating factor. Moreover, antigen‐specific cytotoxicity was demonstrable against: (1) Iad‐bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K‐ras p21 oncogene encoding the corresponding point mutation. CD4+‐mediated cytotoxicity was major histocompatibility complex (MHC) class II‐restricted, as revealed by the absence of lysis against MHC class II− P815 targets, inhibition of A20 lysis with anti‐Iad monoclonal antibodies, and induction of lysis against L cell targets transfected with EαAβd. Independent isolation of a second CD4+ V12 line revealed a very similar cytolytic and MHC class II‐restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4+ Th1 response that specifically recognized tumor cells expressing endogenous activated K‐ras epitopes, which may have implications for the development of peptide‐based active immunotherapies.
Quantitation of endogenous mouse mammary tumor virus superantigen expression by lymphocyte subsets
Authors: Waanders Gary A. , Lees Rosemary K. , Held Werner , MacDonald H. Robson
Published: March 2007

Superantigens (SAg) encoded by endogenous mouse mammary tumor viruses (Mtv) interact with the Vβ domain of the T cell receptor (TcR‐Vβ). Presentation of Mtv SAg can lead to stimulation and/or deletion of the reactive T cells, but little is known about the quantitative aspects of SAg presentation. Although monoclonal antibodies have been raised against Mtv SAg, they have not been useful in quantitating SAg protein, which is present in very low amounts in normal cells. Alternative attempts to quantitate Mtv SAg mRNA expression are complicated by the fact that Mtv transcription occurs from multiple loci and in different overlapping reading frames. In this report we describe a novel competitive polymerase chain reaction assay which allows the locus‐specific quantitation of SAg expression at the mRNA level in lymphocyte subsets from mouse strains with multiple endogenous Mtv loci. In B cells as well as T cells (CD4+ or CD8+), Mtv‐6 SAg is expressed at the highest levels, followed by Mtv‐7 SAg and (to a much lesser extent) Mtv‐8,9. Consistent with functional Mtv‐7 SAg presentation studies, we find that Mtv‐7 SAg expression is higher in B cells than in CD8+ T cells and very low in the CD4+ subset. The overall hierarchy in Mtv SAg expression (i.e. Mtv‐6 > Mtv‐7 > Mtv 8,9) was also observed for mRNA isolated from neonatal thymus. Furthermore, the kinetics of intrathymic deletion of the corresponding TcR‐Vβ domains during ontogeny correlated with the levels of Mtv SAg expression. Collectively our data suggest that T cell responses to Mtv SAg are largely controlled by SAg expression levels on presenting cells.
Transplantation tolerance induced by antigen pretreatment and depleting anti‐CD4 antibody depends on CD4+ T cell regulation during the induction phase of the response
Authors: Bushell Andrew , Morris Peter J. , Wood Kathryn J.
Published: March 2007

Adult mice pretreated with donor‐specific transfusion and depleting anti‐CD4 antibody 28 days before transplant accept fully allogeneic heart grafts and become specifically tolerant without further treatment. The induction of tolerance in this model is not simply a function of CD4+ T cell ablation, but appears to depend on residual CD4+ T cells which escape depletion and engage donor alloantigen during a transient period of antibody blockade. To test the hypothesis that these CD4+ T cells might be responsible for regulating immune responses toward the graft, mice were reconstituted with naive recipient leukocytes at various times after pretreatment. Reconstitution either shortly after pretreatment or shortly after transplant had little effect on graft survival. However, when pretreated mice were given an additional dose of depleting anti‐CD4 antibody at the time of transplant to target putative regulatory cells, naive leukocytes were able to cause acute graft rejection. These data suggest that in clinical transplantation specific T cell regulation might develop following pretreatment with antigen and non‐depleting anti‐CD4 antibodies. Such an approach could provide donor‐specific unresponsiveness prior to transplant without the risks associated with sustained CD4+ T cell depletion.
Polyinosinic acid: polycytidylic acid promotes T helper type 1‐specific immune responses by stimulating macrophage production of interferon‐α and interleukin‐12
Authors: Manetti Roberto , Annunziato Francesco , Tomasevic Ljiljana , Giannò Valeria , Parronchi Paola , Romagnani Sergio , Maggi Enrico
Published: March 2007

The effects exerted on the development in vitro of Dermatophagoides pteronyssinus group I (Der p I)‐specific T cell lines and T cell clones by addition of polyinosinic acid: polycytidylic acid (poly I:C) in lymphocyte bulk culture were examined. Der p I‐specific T cell lines generated in presence of poly I:C exhibited reduced ability to produce interleukin (IL)‐4 and IL‐5 and developed into Der p I‐specific CD4+ T cell clones showing a T helper (Th) type 0 or Th1, instead of Th0/Th2 cytokine profile. This effect was prevented by addition to lymphocyte bulk cultures of a mixture of antibodies specific for interferon (IFN)‐α and IL‐12, whereas the addition of anti‐IFN‐α or anti‐IL‐12 antibody alone was uneffective. Poly I:C also showed the ability to stimulate the production of noticeable amounts of both IFN‐α and IL‐12 by human monocytes. Taken together, these data suggest that poly I:C is a Th1‐inducing agent whose activity is mediated by its ability to stimulate the production of IFN‐α and IL‐12 by monocytes.