bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonspecific augmentation of lymph node T cells and I‐E‐independent selective deletion of VβbTl4+ T cells by Mtv‐2 in the DDD mouse
Authors: Nakano Hideki , Yoshimoto Takayuki , Kakiuchi Terutaka , Matsuzawa Akio
Published: March 2007

DDD/1 (DDD) mice were characterized by marked paucity of T cells in lymph nodes (LN). In DDD‐Mtv‐2/Mtv‐2 (DDD‐Mtv‐2) congenics, T cells were 4‐ to 18‐fold increased depending on ages but B cells doubled at the most. Thymus weight also increased. In DDDfDDD‐Mtv‐2, DDD neonatally infected with Mtv‐2‐derived exogenous MMTV (MMTV‐2), neither LN cells nor thymus weight increased. The Vβ 5+ and Vβ 8+ Tcell contents in LN were practically the same among three strains. The Mtv‐2‐induced expansion of LN Tcells was polyclonal and appeared indigenous to DDD mice. Both Mtv‐2 and MMTV‐2 induced progressive age‐dependent deletion of Vβ 14+CD4+ LN cells. Mtv‐2 but not MMTV‐2 caused deletion of Vβ 14+CD8+ LN cells and mature Vβ 14+CD4+ thymocytes. Thus, Mtv‐2‐ and MMTV‐2‐induced Vβ 14+ T cell deletion may reflect intrathymic and peripheral elimination, respectively. The absence of I‐E gene expression in DDD indicates that Vβ 14+ T cell deletion advances independently of I‐E molecules in this experimental system.
Evidence for programmed cell death of self‐reactive γδ T cell receptor‐positive thymocytes
Authors: Dent Alexander L. , Matis Louis A. , Bluestone Jeffrey A. , Hedrick Stephen M.
Published: March 2007

The negative selection of T cells expressing the γδ T cell antigen receptor (γδ T cells) was studied using transgenic mice expressing a γδ receptor with specificity for an H‐2T‐linked class I major histocompatibility complex molecule from H‐2b mice. The potentially self‐reactive γδ thymocytes in H‐2b/d transgenic mice are larger and have lower levels of γδ T cell receptor expression than γδ thymocytes from H‐2d mice. H‐2b/d γδ thymocytes do not respond to H‐2b antigen‐presenting cells, and thus are inactive compared to H‐2d γδ thymocytes. However, the H‐2b/d γδ thymocyte population, but not the H‐2d γδ thymocyte population, undergoes a high rate of programmed cell death when placed in overnight culture. These observations constitute the first direct evidence that self‐reactive γδ thymocytes undergo programmed cell death. This in vitro programmed cell death of self‐reactive γδ thymocytes may reflect the clonal deletion process that results in a depletion of γδ T cells in the peripheral lymphoid organs of adult H‐2b/d mice. We also present evidence that self‐reactive γδ T cells, similarly to αβ T cells, undergo a lesser degree of clonal deletion in neonatal mice compared to adult mice.
In vivo administration of major histocompatibility complex class I‐specific peptides from influenza virus induces specific cytotoxic T cell hyporesponsiveness
Authors: Müllbacher Arno , Hla Ron Tha
Published: March 2007

We have been investigating the immunogenicity of two class I major histocompatibility complex‐specific peptides with a sequence derived from influenza virus nucleoprotein specific for Kd and one for Db. Peptide‐modified splenocytes are unable to immunize for a primary cytotoxic T (Tc) cell response in vivo, or secondary response in vitro. Peptide‐modified stimulator cells can boost virus‐primed splenocytes for a strong secondary response in vitro. Animals primed with syngeneic peptide‐modified splenocytes upon challenge with virus in vivo do not generate strong secondary Tc cell responses on day 3 after challenge in contrast to virus primed animals. Day 6 responses of virus‐challenged, peptide‐primed animals are reduced as compared to unprimed mice. This hyporesponsiveness is independent of CD8+ T cells in the priming population and can be elicited with tumor cell lines. The data are discussed in the framework of the two‐signal model of immune induction.
The G protein‐coupled receptor BLR1 is involved in murine B cell differentiation and is also expressed in neuronal tissues
Authors: Kaiser Edelgard , Förster Reinhold , Wolf Ingrid , Ebensperger Cecilia , Michael Kuehl W. , Lipp Martin
Published: March 2007

The BLR1 gene, isolated initially from Burkitt lymphoma cells (Eur. J. Immunol. 1992. 22:2795), encodes a G protein‐coupled receptor with significant relationship to receptors for chemokines (IL‐8, MIP‐1α) and neuropeptides. The murine homologue of human BLR1 was cloned and used to investigate its expression in vivo. Brl1‐specific transcripts are observed in secondary lymphatic organs and to a lesser extent in brain of adult mice but not in other tissues. RNA in situ hybridization localizes blr1 transcription to primary follicles and to the mantle zone of secondary follicles. SCID mice in which mature B cell development is severely impaired exhibit a strongly reduced level of blr1‐specific RNA in the spleen. The analysis of murine lymphoid tumor cell lines representing distinct stages of the B cell lineage reveals elevated expression of blr1 in B cell lymphomas but not in pre‐B lymphomas or plasmacytomas. Induction of differentiation of resting B cells by cytokines or mitogens down‐regulates expression of blr1. RNA in situ hybridization using brain sections of adult mice detects blr1 transcription in the granule and Purkinje cell layer of the cerebellum. Interestingly, the blr1 gene is also expressed during late embryogenesis in fetal liver and brain. In view of the remarkable expression pattern in the B cell lineage we suggest that murine BLR1 may represent a cytokine/neuropeptide receptor exerting regulatory functions on recirculating mature B lymphocytes.
Characterization of host CD4+ T lymphocytes in mice neonatally tolerized to alloantigens
Authors: Kramar Giulia , Aguado Teresa , Louis Jacques , Del Giudice Giuseppe , Lambert Paul‐Henri
Published: March 2007

BALB/c mice injected at birth with semi‐allogeneic F1 spleen cells become tolerant to alloantigens as shown by their CTL unresponsiveness to the corresponding alloantigen and the persistence of donor F1 cells into the BALB/c host. Moreover, these mice develop a transient systemic lupus erythematosis‐like autoimmune syndrome characterized by splenomegaly, glomerulonephritis, thrombocytopenia and abnormal serological findings, such as several autoantibodies and IgG1 hypergammaglobulinemia. Recent studies done in our laboratory have shown that donor F1 B cells persisting in the host are responsible for the production of autoantibodies and must be activated in vivo by the host CD4+ T lymphocytes in a MHC class II‐restricted fashion. In the present work, we have focused our attention on the ability of splenic CD4+ T cells recovered at different periods from BALB/c mice injected at birth with (CBA/Ca × BALB. Ighb) Fl spleen cells to interact with and activate F1 semi‐allogeneic spleen cells in vitro. We show that (i) only CD4+ T cells from 2‐ and 3‐week‐old tolerant BALB/c mice preferentially produce IL‐4 and IL‐5 in response to a F1 semi‐allogeneic in vitro stimulation, (ii) CD4+ T cells purified from 3‐week‐old tolerant BALB/c mice are able to induce in vitro IgG and IgM production by F1 B cells. Taken together, these results strongly suggest that host CD4+ T cells, belonging to the TH2 subset progressively lose their reactivity towards the F1 semi‐allogeneic persistent B cells, reaching a state of unresponsiveness that correlates with the disappearance of serum autoantibodies and autoimmune pathology.
Protein tyrosine phosphorylation is mandatory for CD40‐mediated rescue of germinal center B cells from apoptosis
Authors: Knox Kirstine A. , Gordon John
Published: March 2007

Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross‐linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose‐dependent fashion, by pre‐treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti‐CD40 was remarkably similar to that triggered by polyvalent anti‐Ig. By contrast, anti‐CD40 failed to stimulate the increase in inositol 1,4,5‐trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca2+. Pre‐incubation with the PTK inhibitor herbimycin A (5 μM) abrogated anti‐CD40‐mediated rescue of GC B cells from apoptosis, while genistein (40 μM) and the tyrphostins AG490 (10 μ M) and AG814 (25 μ M) significantly inhibited this process. Consistent with these results, herbimycin A (5 μM) abolished the expression of the 26 kDa bcl‐2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti‐CD40. The Ca2+ chelators BAPTA and EGTA did not significantly affect CD40‐promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40‐mediated rescue of GC B cells from apoptosis.
Mitogenicity of anti‐Thy‐1 monoclonal antibodies attributable to an Fc‐dependent mechanism
Authors: Kummer Udo , Haunschild Jutta , Reisbach Gilbert , Delecluse Henri‐Jacques , Thierfelder Stefan
Published: March 2007

We analyzed the mechanism by which certain anti‐Thy‐1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti‐Thy‐1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico‐chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy‐1 molecules, following specific interactions with anti‐Thy‐1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy‐1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self‐associate, probably through homophilic Fc‐Fc contacts, and that this feature renders anti‐Thy‐1 mAb mitogenic. To prove this, we set up cross‐inhibition studies with randomly selected mitogenic (IgG2c) and non‐mitogenic (IgG2b) anti‐Thy‐1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c‐derived F(ab')2 fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell‐bound IgG2c mAb with a monomeric and thus non‐aggregating IgG‐binding protein A‐derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross‐linking via FcγR. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy‐1 molecules, thereby stimulating the mitotic apparatus of the cell.
Th1 clones that suppress IgG2ab specifically recognize an allopeptide determinant comprising residues 435–451 of γ2ab
Authors: Bartnes Kristian , Rekdal Øystein , Briand Jean‐Paul , Hannestad Kristian
Published: March 2007

We have previously reported that γ2ab/I‐Ad‐specific Th1 clones from BALB/c mice (γ2aa, H‐2d) mediated a long‐lasting, selective suppression of serum IgG2ab levels when transferred to newborn (BALB/c X B10.D2)F1 (γ2a/b, H‐2d) mice (Bartnes, K. and Hannestad, K. Eur. J. Immunol. 1991. 21: 2365). We here analyze the peptide specificity of hybridomas derived from two suppressive T cell clones. The shortest synthetic peptide with optimal antigenicity comprises γ2ab residues 435–451 (Kabat numbering). The determinant core encompasses the γ2ab 440–446 (KLRVQKS) sequence which contains an I‐Ad allele‐specific motif. Challenge with single amino acid‐substituted γ2ab 435–447 analogs revealed that residues K440, R442 and K445 which are shared by the autologous and allogeneic γ2a, as well as residues Q444 and S446 which represent allogeneic differences, are critical for recognition. We obtained evidence that K440, R442 and Q444 are epitope residues, while K445 and S446 contribute to anchoring of the peptide to I‐Ad. Amino acids located outside of the core also influence antigenicity, the most striking effect being a 340–870‐fold augmentation of potency when γ2ab 437–451 is extended by F436. IgG2ab required processing in order to stimulate the hybridomas. The data support the contention that the Th1 clones specific for Fc of γ2ab mediated IgG2ab suppression by cognate interaction with sIgG2ab+ B cells that presented a Cγ2ab peptide(s) derived from their endogenous Ig on major histocompatibility complex class II. The T cells cross‐reacted weakly with peptide 435–451 of the autologous γ2aa allotype. This opens the possibility that self‐peptides from Ig C regions can target B cells for regulatory interactions with autologous Th cells.
Complementarity‐determining region 2 is implicated in the binding of staphylococcal protein A to human immunoglobulin VHIII variable regions
Authors: Randen Ingrid , Potter Kathleen N. , Li Yucheng , Thompson Keith M. , Pascual Virginia , Fθrre Østein , Natvig Jacob B. , Donald Capra J.
Published: March 2007

Staphylococcal protein A (SPA) has two distinct binding sites on human immunoglobulins. In addition to binding to the Fc region of most IgG molecules, an “alternative” binding site has been localized to the Fab region of human immunoglobulins encoded by heavy chain variable gene segments belonging to the VHIH family. Comparison of amino acid sequences of closely related SPA‐binding and ‐non‐binding proteins suggested that VHIII‐specific residues in the second complementarity‐determining region (CDR2) were likely responsible for SPA binding activity. Site‐directed mutagenesis of a single amino acid residue in CDR2 converted an IgM rheumatoid factor which did not bind SPA to an SPA binder. These findings, therefore, locate a critical site involved in SPA binding to the CDR2 of human immunoglobulins encoded by VHIII family gene segments.
Overlapping epitopes encompassing a point mutation (12 Gly → Arg) in p21 ras can be recognized by HLA‐DR, ‐DP and ‐DQ restricted T cells
Authors: Fossum Beate , Gedde‐Dahl Tobias , Hansen Torbjern , Eriksen Jon Amund , And Erik Thorsby , Gaudernack Gustav
Published: March 2007

Mutations in ras genes which result in transforming gene products carrying amino acid substitutions in position 12, 13 or 61 are common in human cancer. Peptides encompassing these mutations in ras are shown to be immunogenic in both mice and humans. The potential usefulness of such peptides in cancer therapy, depends on their ability to bind to HLA molecules. We therefore stimulated T cells from healthy donors with mutated ras‐derived peptides. By repeated in vitro stimulation of peripheral blood mononuclear cells, several T cells clones could be generated which recognized a p21 ras derived peptide carrying a position 12 Gly → Arg substitution. This peptide (1–25, 12 Arg) could be specifically recognized by T cells restricted by either HLA‐DQ7 or ‐DP3. Previously, we showed that this peptide is also recognized by a T cell clone restricted by HLA‐DR2. The core region of the peptide was determined to span positions 9–16 for all three HLA restriction elements, and accordingly contains the mutational hot spots in position 12 and 13. The observation that the mutant 1–25, 12 Arg ras‐derived peptide may contain a promiscuous epitope encompassing the Gly → Arg mutation in position 12 indicates that lack of peptide presentation by given HLA molecules may not be a major constraint in responsiveness against ras mutations.
Number of interleukin‐4‐ and interferon‐γ‐secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation
Authors: Elghazali Gehad E. B. , Paulie Staffan , Andersson Gudrun , Hansson Yngve , Holmquist Göran , Sun Jia‐Bin , Olsson Tomas , Ekre Hans Peter , Troye‐Blomberg Marita
Published: March 2007

The enzyme‐linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin‐4 (IL‐4)‐ and interferon‐γ (IFN‐γ)‐producing cells in in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA‐induced IL‐4 and IFN‐γ secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN‐γ‐ and very few IL‐4‐producing cells, while TT‐induced both IL‐4 and IFN‐γ. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell‐mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody‐dependent, Th 2 type of response.

In individuals recently boosted with TT, a significant increase in both IL‐4‐ and IFN‐γ‐producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN‐γ‐producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen‐specific and nonspecific T cells are activated and that antigen‐specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell‐bound or soluble receptors.
An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV‐1 envelope glycoprotein
Authors: Bergmann Cornelia , Stohlmann Stephen A. , McMillan Minnie
Published: March 2007

The immunodominant H‐2Dd‐restricted cytotoxic T lymphocyte (CTL) response to the HIV‐1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320‐327). However, 9mer and l0mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing Dd‐expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10‐amino acid determinant (residues 318‐327) and a 67‐amino acid peptide (residues 281‐348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild‐type gpl60 in their ability to sensitize target cells for lysis by gpl60‐specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160‐specific CTL. These data demonstrate that both the V3 loop itself and a 10‐residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene‐encoded CTL epitopes for recombinant vaccines designed to induce protective T cell‐mediated immunity against HIV‐1.
Characterization of two new CD18 alleles causing severe leukocyte adhesion deficiency
Authors: Rodríguez Cristina Löpez , Nueda Arsenio , Grospierre Barbara , Fischer Alain , Springer Timothy A. , Corbi Angel L.
Published: March 2007

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by heterogeneous mutations within the gene encoding the common β subunit (CD18) of the three leukocyte integrins LFA‐1 (CD11a/CD18), Mac‐1 (CD11b/CD18), and pl50,95 (CD11c/CD18). Based on the level of expression of CD18 on patient leukocytes, two phenotypes of LAD have been defined (severe and moderate) which correlate with the severity of the disease. We have investigated the molecular basis of the disease in two unrelated severe patients (HS and ZJO). Both patients share a complete absence of CD18 protein precursor and cell surface expression, but they differ in the level of CD18 mRNA, which is normal in HS and undetectable by Northern blot in ZJO. Determination of the primary structure of the patient HS CD18 mRNA revealed a 10‐base pair deletion between nucleotides 190‐200 (CD 18 exon 3), which eliminates residues 41–43 and causes a frameshift into a premature termination codon 17 base pairs downstream from the deleted region. The 10‐base pair frameshift deletion maps to a region of the CD18 gene where aberrant mRNA processing has been detected in HS and two other unrelated LAD patients. In the ZJO patient, amplification of lymphoblast CD 18 mRNA demonstrated the presence of a non‐sense mutation in the third nucleotide of the triplet encoding Cys534 (TGC → TGA), within exon 12. Both genetic abnormalities were also detected at the genomic level, and affect the restriction pattern of their corresponding genes, thus enabling the detection of the mutant alleles among healthy heterozygous alleles in family studies. The identification of two new LAD CD18 alleles, either carrying a non‐sense mutation (ZJO) or a partial gene deletion (HS), further illustrates the heterogeneity of the genetic alterations in LAD.
ICAM‐3, the third LFA‐1 counterreceptor, is a co‐stimulatory molecule for both resting and activated T lymphocytes
Authors: Hernandez‐Caselles Trinidad , Rubio Gonzalo , Campanero Miguel R. , del Pozo Miguel Angel , Muro M. , Sanchez‐Madrid Francisco , Aparicio Pedro
Published: March 2007

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co‐stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter‐receptors on antigen‐presenting cells. Soluble ICAM‐3, anti‐ICAM‐3 and anti‐CD3 mAb were utilized to address the role of the ICAM‐3/LFA‐1 pathway in TcR/CD3‐dependent or ‐independent T cell activation. Immunoaffinity‐purified ICAM‐3 co‐immobilized with suboptimal concentrations of anti‐CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM‐3 with a β2 integrin, likely to be LFA‐1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti‐ICAM‐3 mAb were able to co‐stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti‐ICAM‐1 mAb were only co‐stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM‐3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti‐ICAM‐3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM‐3 on the surface of both T cells and antigen‐presenting cells plays an essential role in the initiation of the immune response.
Schistosoma mansoni circulating anodic antigen but not circulating cathodic antigen interacts with complement component C1q
Authors: van Dam Govert J. , Seino Jin , Rotmans J. Peter , Daha Mohamed R. , Deelder André M.
Published: March 2007

Adult schistosome parasites, living in the blood vessels of their mammalian hosts, protect themselves against immune damage in a variety of ways. In addition to the tegument, the intestinal epithelium of the blood‐feeding worms is permanently exposed to both the innate and the acquired immune system. In this study, we investigated whether the Schistosoma gut‐associated antigens CAA and CCA (circulating anodic antigen and circulating cathodic antigen, respectively), which are excreted in relatively large quantities into the host's circulation, might play a role in evading complement attack. Of several complement components tested, only purified C1q showed significant binding to CAA, a negatively charged highly glycosylated glycoprotein. CCA, also highly glycosylated, but neutral or slightly positively charged, did not bind to C1q. CAA bound only to the collagen‐like stalks of C1q and not to the globular heads. No detectable interaction of CAA with precursor human CI was found and CAA did not induce activation of CI in whole human serum as assessed by consumption of hemolytic C4 activity. Also CAA could not induce activation of precursor CI in vitro. These results suggest that CAA behaves like a receptor for C1q, and might be involved in protecting the vulnerable schistosome gut against complement‐mediated attack.
Signaling of programmed cell death induction in WEHI‐231 B lymphoma cells
Authors: Hibner Urszula , Benhamou Laure E. , Haury Matthias , Cazenave Pierre‐André , Sarthou Pierre
Published: March 2007

The murine WEHI‐231 B lymphoma is highly sensitive to membrane immunoglobulin ligation which leads to programmed cell death (PCD) in this cell line. To study the molecular pathways involved in PCD induction in these cells, we derived two variants of WEHI‐231 resistant to anti‐Ig treatment. The level of bcl‐2 mRNA was identical in the wild type and the variants, either untreated or anti‐Ig treated, suggesting that PCD is not under the control of bcl‐2 in WEHI‐231 cells. In contrast, c‐myc gene expression was markedly different in the wild type and the variants, both in the unstimulated and anti‐Ig‐stimulated state. Our findings are interpreted in the context of the dual capacity of c‐myc to promote cell growth or cell death, in conjunction with other growth regulatory signals.
Cross‐linking of CD59 and of other glycosyl phosphatidylinositol‐anchored molecules on neutrophils triggers cell activation via tyrosine kinase
Authors: Morgan B. Paul , van den Berg Carmen W. , Davies Eryl V. , Hallett Maurice B. , Horejsi Vaclav
Published: March 2007

Many membrane proteins are attached via a glycosyl phosphatidylinositol (GPI) anchor. Proteins anchored in this way make no direct contact with the interior of the cell, therefore a role in signaling or activation would seem unlikely. Nevertheless, cross‐linking of GPI‐anchored proteins on human and murine T lymphocytes has been shown to cause calcium transients and cell activation. Our studies address the non‐lethal events caused by the membrane attack complex of complement, which include release of Ca2+ from intracellular stores, and have suggested that the GPI‐anchored complement inhibitor CD59 may be involved in signaling these events. We here report that cross‐linking of CD59 on human neutrophils using specific monoclonal antibody and second antibody caused a rapid increase in intracellular free Ca2+ concentration (Ca2+ transient) due to release of Ca2+ from stores and also caused neutrophil oxidase activation. All antibodies against CD59 tested were effective and cross‐linking of any other GPI‐anchored protein expressed on neutrophils also initiated an increase in intracellular free Ca2+ concentration, whereas cross‐linking of transmembrane proteins caused little or no response. A tyrosine kinase‐dependent activation pathway was indicated by the demonstration of tyrosine phosphorylation on cross‐linking and by blocking of the Ca2+ transient with the tyrosine kinase inhibitor herbimycin.
Patterns of major histocompatibility complex class II expression on T cell subsets in different immunological compartments 1. Expression on resting T cells
Authors: Dutia Bernadette M. , McConnell Ian , Bird Kay , Keating Paula , Hopkins John
Published: March 2007

In this study we have investigated the expression of major histocompatibility complex (MHC) class II molecules on T cells from various lymphoid compartments in the sheep. Monoclonal antibodies which react specifically with sheep MHC class II molecules homologous to the human DQ and DR molecules have been characterized. These antibodies have been used, together with the monoclonal antibodies specific for sheep CD4‐, CD8‐ and T19‐positiveT cells, to quantitate DQ and DR expression on T cell subsets in adult and fetal peripheral blood, afferent lymph, lymph node and efferent lymph. The results show that expression of class II by T cells depends on the age of the animal and the physiological location of the T cell. In fetal blood there is no expression of class II on CD8+ or T19+ cells and very low expression on CD4+ T cells. In adult peripheral blood and efferent lymph a significant proportion of cells express DR but not DQ. A very different situation is found in afferent lymph and the peripheral lymph node: in afferent lymph the majority of T cells in all three subsets express both DQ and DR molecules; in the lymph node over 50% of T cells express DR and 30 % are DQ+. These results suggest that within all T cell subsets there is a progression from DQ− DR− to DQ−DR+ and DQ+DR+ which correlates with physiological stages of T cell differentiation in vivo.
Major histocompatibility complex regulation of interleukin‐5 production in the mouse
Authors: Dieli Francesco , Sireci Guido , Lio Domenico , Bonanno Cesira T. , Salerno Alfredo
Published: March 2007

Lymph node cells of CBA (H‐2k), but not of BALB/c (H‐2d) mice immunized epicutaneously with picryl chloryde secrete interleukin (IL)‐5 when stimulated with the specific antigen in vitro. The low IL‐5 production in BALB/c mice persists when either picryl chloride or the unrelated antigen oxazolone are used, when the amount of antigen in vitro is varied and when a secondary response is studied. The difference in IL‐5 production maps to the major histocompatibility complex (MHC) in the congenic BALB/b, BALB/c and BALB/k mice. Furthermore, lymph node cells from (k × d) F1 mice produce IL‐5 when stimulated by antigen presented on H‐2k but not on H‐2d antigen‐presenting cells.

Finally, the low IL‐5 production in vitro in BALB/c mice is correlated with low picryl‐specific IgA levels in vivo, which otherwise are ten times greater in CBA and BALB/k mice. The influence of MHC on IL‐5 production and IgA secretion in the mouse might be a possible basis for the association of MHC with IgA deficiency in humans.
The thymus‐independent antigen α(1–3) dextran elicits proliferation of precursors for specific IgM antibody‐producing cells (memory cells), which are revealed by LPS stimulation in soft agar cultures and detected by immunoblot
Authors: Kolb Cornelia , Fuchs Bergund , Weiler Eberhardt
Published: March 2007

Single antibody‐forming cells (AFC) specific for α(l‐3) dextran (Dex) from i.p.‐immunized BALB/c mice were enumerated in soft agar cultures by blotting on antigen‐precoated membranes and subsequent staining via enzyme‐coupled anti‐IgM antibodies. Short cultures (2 h) revealed AFC as harvested ex vivo, while in long‐term cultures (4 days), in the presence of lipopolysaccharide (LPS) as B cell mitogen, cells or colonies developed by differentiation in vitro. Whereas the spleen contained most AFC ex vivo in a sharp‐peak response at 4 and 5 days after i.p. injection of Dex in aqueous solution, peritoneal exudate cells (PEC) contained only very few AFC. However, the same PEC population developed Dex‐specific cells or colonies after 4 days of culture. The isotype of antibodies was IgM. The frequency of these Dex‐specific LPS‐inducible precursor cells rose exponentially in the course of the immune response to a broad plateau and was still, 11 weeks after Dex injection, approximately 40‐fold higher than in non‐immunized mice.

Since these cells increased in frequency after antigen injection, and since they could not be detected as AFC during 2 h ex vivo, they were regarded as memory cells. They seemed to be arrested in vivo, but could be induced to differentiation and/or proliferation in vitro. Although these cells had the functional characteristics of memory cells as defined above, they produced anti‐Dex antibodies of IgM isotype. Their population might be critical for the protection of the peritoneal cavity against microbial invasion from the intestines, and it may be significant in this context that we could evoke a peritoneal memory cell response only when antigen was injected intraperitoneally, but not intravenously. In athymic BALB/c‐nu/nu mice only few of these Dex‐specific memory cells were found. It is possible that T cells exert a regulatory influence on this pathway of differentiation.
Differences in the requirement of antigen‐presenting cells for long‐term growth among cytomegalovirus‐specific Th clonotypes
Authors: Santamaria Pere , Lewis Cinthia , Barbosa Jose J.
Published: March 2007

Functional and molecular studies of in vivo‐activated T lymphocytes involved in normal and abnormal immune responses, i. e., cells infiltrating tissues affected by autoimmune processes, require their previous in vitro expansion. Problems such as unavailability of specific antigen(s) (Ag) and/or the requirement of large amounts of autologous peripheral blood mononuclear cells (PBMC) as feeders have prompted the development of alternative expansion methods that circumvent the use of antigen‐presenting cells (APC) and/or Ag. We have previously shown that cytomegalovirus (CMV)‐specific Tcell lines and clones can be efficiently propagated in long‐term cultures by stimulation with agonistic monoclonal antibodies (mAb) coated onto polystyrene particles in the absence of APC. However, this and other stimulation protocols may skew the repertoire of clonotypes that proliferate in response to nominal Ag and APC. Here we show that polyclonal populations of CMV‐primed and interleukin‐2 (IL‐2)‐stimulated PBMC undergo the same clonotypic selection when induced to grow both by continuous stimulation with CMV and an anti‐CD3 mAb presented by APC. This selection, reproduced in three independent expansion experiments, involved the dominant growth of two CMV‐specific, IL‐2‐secreting CD4+ clonotypes sharing Jα, Jβ, Vα and Vβ genes and highly homologous T cell receptor (TcR) junctional sequences. The dominant growth of these 2 clonotypes required a direct Tcell/APC interaction since when coated onto polystyrene particles the same mAb induced the selective expansion of another IL‐2‐secreting CD4+ CMV‐specific clonotype expressing a different, yet homologous, TcR heterodimer (used same Vα and Vβ genes), which was underrepresented before expansion (5 vs. 58%). Tcell clones belonging to the subdominant clonotype proliferated significantly faster in response to stimulation with anti‐CD3 mAb coated onto beads than in response to stimulation with CMV or anti‐CD3 mAb presented by APC, possibly due to long‐term expansion without APC or antigen. In contrast, neither the dominant clonotypes nor unprimed T cells were able to undergo CD3‐mediated expansion in the absence of APC. We conclude that quantitative differences in growth competency among nominal Ag‐activated T‐helper (Th) clonotypes in response to antigenic stimulation can be reproduced by stimulation through the TcR in the absence of TcR occupancy but only in the presence of APC and that certain clonotypes do not require APC for long‐term growth in vitro. These data have practical implications for the isolation and repertoire characterization of in vivo‐activated T cells from tissues affected by inflammatory, i.e. autoimmune, phenomena.
Selective coupling of the T cell antigen receptor to phosphoinositide‐derived diacylglycerol production in HPB‐ALL T cells correlates with CD45‐regulated p59fyn activity
Authors: Biffen Mark , Shiroo Masahiro , Alexander Denis R.
Published: March 2007

In HPB‐ALL T‐cells the p59fyn tyrosine kinase is regulated by the CD45 phosphotyrosine phosphatase and plays a critical role in coupling the T cell receptor (TCR) to the generation of intracellular signals which include diacylglycerol (DAG) production and protein kinase C activation. The aim of this study was to determine the phospholipid pools from which the DAG is generated and to identify which phospholipase activities are regulated by the TCR. When CD45+cells were pre‐labeled with [3H] arachidonic acid, CD3‐antigen cross‐linking stimulated negligible increases in both [3H]DAG and [3H] phosphatidic acid (PA). However, CD3 monoclonal antibody (mAb) induced an increase of 300 % in [3H]PA when the cells were permeabilized with streptolysin‐O, and this correlated with increased levels of protein tyrosine phosphorylation. Stimulation of [3H] PA production upon CD3 cross‐linking was 77 % lower in permeabilized CD45− cells than in CD45+ cells, consistent with the reduced activity of p59fyn in CD45− cells. The stimulated production of PA was not mediated by activation of phospholipase D (PLD), although the presence of a G‐protein‐regulated PLD activity was established. The CD3‐induced increase in total inositol phosphates (InsP) in permeabilized cells was similar to the stimulated production of [3H] PA production in both CD45+ and CD45− cells. Dose‐response curves for InsP and PA production triggered by CD3 mAb were super‐imposable and the production of InsP and PA over a range of Ca2+ concentrations was comparable. Differential labeling of phospholipids with 3H‐labeled fatty acids revealed that CD3‐induced PA production reflected incorporation of label into the phosphatidylinositol pool. Our data suggest that in HPB‐ALL cells the production of DAG following CD3‐antigen cross‐linking can be fully accounted for by the selective coupling of the TCR to breakdown of phosphatidylinositol‐(4,5)‐bisphosphate as the result of phospholipase Cγ1 activation. This event correlates with the activity of the CD45‐regulated TCR‐associated tyrosine kinase, p59fyn.
Transcriptional regulation of interleukin‐2 gene expression by CD69‐generated signals
Authors: D'Ambrosio Daniele , Trotta Rossana , Vacca Alessandra , Frati Luigi , Santoni Angela , Gulino Alberto , Testi Roberto
Published: March 2007

The 5′ flanking region of the human interleukin (IL)‐2 gene was investigated for enhancer activity in response to CD69‐generated signals, using a chloramphenicol acetyltransferase (CAT)‐driven transient expression system in Jurkat cells. The region extending from −317 to +47 relative to the initiation site of IL‐2 gene transcription was shown to contain sequences able to respond to CD69 cross‐linking, by enhancing by about 100 % a phorbol 12‐myristate 13‐acetate (PMA)‐plus‐ionomycin stimulation of CAT activity. A similar increase in CAT activity produced by PMA‐plus‐anti‐CD3 mAb was induced by CD69 cross‐linking, while a 200 % increase over that obtained by PMA‐plus‐anti‐CD28 mAb stimulation was seen. Analysis of enhancer deletion mutants revealed that proximal AP‐1, OCT‐1/octamer‐associated protein and nuclear factor of activated T cells (NFAT) binding regions were all necessary to allow CD69‐mediated enhancement of CAT activity. By gel mobility shift analysis, cyclosporin A‐sensitive NFAT‐binding induction and enhancement of AP‐1 binding activity could be detected in nuclear extracts of both Jurkat and peripheral blood T cells after simultaneous CD69 and protein kinase C stimulation. Finally, CD69‐mediated signals could increase NFAT and AP‐1 binding activity following PMA and ionomycin stimulation in peripheral blood T cells. Collectively, these data suggest that CD69‐generated signals participate in the control of the IL‐2 gene expression at the transcriptional level, likely acting through NFAT and AP‐1 transcription factor complexes.
A soluble form of the adhesion receptor CD58 (LFA‐3) is present in human body fluids
Authors: Hoffmann Jörg C. , Dengler Thomas J. , Knolle Percy A. , Albert‐Wolf Marion , Roux Matthias , Wallich Reinhard , Meuer Stefan C.
Published: March 2007

The human adhesion receptor CD58 (LFA‐3) is expressed on most human cell types. Here we report on a soluble form of CD58 (sCD58) in human serum, human urine, and culture supernatants of several cell lines. sCD58 partially purified from human serum, from supernatant of the Hodgkin cell line L428, and purified sCD58 from human urine were found to have a molecular mass of 40‐70 kDa under denaturating conditions (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting). However, gel filtration of sCD58 purified from human urine gave a molecular mass of 118‐166 kDa, suggesting a noncovalent homotrimer conformation or its association with other molecules. Using an enzyme‐linked immunosorbent assay specific for CD58 we found that sera from patients suffering from different forms of hepatitis contained elevated sCD58 levels (n = 108). Accordingly, there was a fivefold increase of supernatant sCD58 when the hepatocellular carcinoma cell line Hep G2 was incubated with 25 ng/ml recombinant tumor necrosis factor‐α in vitro. In contrast, sCD58 serum levels of 337 additional patients suffering from various other immunological disorders were not found to be raised. At high concentrations sCD58 binds to CD2‐positive cells and inhibits rosette formation of human T cells to human erythrocytes. Thus, local release of large quantities of naturally occurring sCD58 may interfere with intercellular adhesion in vivo.
Analysis of tetanus toxin peptide/DR recognition by human T cell receptors reconstituted into a murine T cell hybridoma
Authors: Blank Ulrich , Boitel Brigitte , Mège Dominique , Ermonval Myriam , Acuto Oreste
Published: March 2007

We have previously reported that human T cell receptors (TcR) selected in the class II‐restricted (HLA‐DRB1*1302) response to a tetanus toxin peptide (tt830‐843) frequently used the Vβ2 germ‐line segment which paired with several Vα segments and that the putative CDR3 of both α and β chains showed remarkable heterogeneity. To analyze the structural basis for recognition of the tt830‐843/DR complex, five of these TcR were reconstituted into a murine T cell hybridoma, 58 α−β−, by expressing the human α and β variable regions joined to the mouse α and β constant regions, respectively. The chimeric TcR, expressing the same Vβ germ‐line segment (Vβ2), two expressing Vα21.1, twoVα17.1 and one Vα8.1 were shown to have the expected antigen specificity and DR restriction.

Two lines of evidence suggested that the putative CDR3, although not conserved in these TcR, played a key role in recognition. First, two TcR with identical V germ‐line segments but distinct CDR3 showed large differences in their capacity to react with the ligand. Second, interchanging the α and β chains from tt830‐843/DR1302‐specific TcR which differed in their CDR3 sequences invariably led to loss of recognition. We also asked whether germ‐line Vα17.1 could functionally replace Vα21.1, as they appear to be related in their primary sequence. However, as in the case of CDR3 exchanges, Vα replacement abrogated TcR reactivity. Taken together, these data underline the fine interdependence of the structural components of the TcR binding site in defining a given specificity.

Four of the TcR studied displaying promiscuous recognition were also tested against different DR alleles and site‐directed mutants. The results of these experiments suggested that, in spite of their structural heterogeneity, anti‐tt830‐843 TcR may have a similar orientation with respect to the peptide/DR complex.

The reconstitution system described herein should represent a valuable tool for detailed studies of human TcR specificity.
Continuous in vivo activation and transient hyporesponsiveness to TcR/CD3 triggering of human gut lamina propria lymphocytes
Authors: de Maria Ruggero , Fais Stefano , Silvestri Maurizio , Frati Luigi , Pallone Francesco , Santoni Angela , Testi Roberto
Published: March 2007

Three‐color immunofluorescence and flow cytometric analysis showed that the vast majority of normal human T‐lamina propria lymphocytes (LPL) expressed high levels of the early activation antigen CD69, together with CD45R0, irrespective of their CD4, CD8 or γ/δ‐TcR phenotype, indicating that they are continuously stimulated in vivo. Importantly, measurement of cytoplasmic [Ca2+]i showed that T‐LPL had significantly higher basal [Ca2+]i levels, compared to autologous peripheral blood lymphocytes (PBL). Both cytoplasmic [Ca2+]i elevation and inositol (1, 4, 5) trisphosphate generation following CD3 cross‐linking by monoclonal antibodies in vitro were essentially abolished in T‐LPL, as compared to autologous T‐PBL. Moreover, freshly isolated LPL could be induced to proliferate by CD2‐ or CD28‐mediated signals, but not by CD3‐mediated signals. Surprisingly however, impairment in TcR/CD3‐mediated early signaling and proliferation in T‐LPL could be completely reversed by 24 h incubation of the cells at 37 °C in culture medium, a condition which allowed basal intracellular [Ca2+]i to return to levels comparable to peripheral T cells.

Our data suggest that selective hyporesponsiveness to TcR/CD3‐mediated signaling may represent a transient event during continuous in vivo activation of mucosal lymphocytes.
The mitogenic response of T cells to interleukin‐2 requires Raf‐1
Authors: Riedel Detlev , Brennscheidt Ulrich , Kiehntopf Michael , Brach Marion , Herrmann Friedhelm
Published: March 2007

The product of the c‐raf‐1 proto‐oncogene, Raf‐1, is known to encode a 74‐kDa ubiquitously expressed cytoplasmic serine/threonine kinase. Various growth factors such as epidermal growth factor, acidic fibroblast growth factor, platelet‐derived growth factor, insulin, granulocyte‐macrophage colony‐stimulating factor, interleukin (IL)‐2, IL‐3 and erythropoietin have been shown to induce phosphorylation of Raf‐1, thereby activating Raf‐1 kinase. Raf‐1 is, thus, believed to play a role in coupling growth factor receptors to proliferation. We have examined the role of Raf‐1 in the mitogenic response of human peripheral blood‐derived IL‐2 receptor expressing T cells to human recombinant IL‐2 employing c‐raf antisense (AS) oligodeoxyribonucleotide. Uptake studies of oligonucleotides indicated that incorporation of oligomers was maximal at 4 h and oligdeoxynucleotides remained stable in these cells for up to 24 h. Treatment of T cells with the AS oligodeoxyribonucleotide in intracellular duplex formation followed by efficient translation blockade of c‐raf‐1. In contrast, sense (S) and nonsense (NS) oligodeoxynucleotides failed to form intracellular duplexes and did not interfere with translation of c‐raf‐1, suggesting specific elimination of c‐raf‐1 by the AS oligomer. Proliferation of T cells ([3H]thymidine incorporation) following exposure to IL‐2 was substantially reduced when the c‐raf‐1 AS oligodeoxyribonucleotide was added to cultures, while the mitogenic response to this factor remained almost unaffected in the presence of S and NS oligodeoxy‐ribonucleotides.