bluuebunny/crossref_metadata_2025 huggingface dataset:
Human immune response to group A streptococcal carbohydrate (A‐CHO) III. Comparison of the efficiencies of anti‐idiotopic antibody and of nominal antigen in the induction of IgM anti‐A‐CHO‐producing B cells
Authors: Bloem Andries C. , Jürgens Rainer , Eichmann Klaus , Emmrich Frank
Published: March 2007

In this study the efficiencies of a monoclonal anti‐idiotopic (Id) antibody (anti‐Id498) and of various preparations of nominal antigen in the induction of an antigen‐specific human B cell response in vitro were compared. Anti‐Id498 recognizes a recurrent, binding site‐related Id present on IgM antibodies with specificity for N‐acetyl‐D‐glucosamine, the immunodominant group of streptococcal group A carbohydrate (A‐CHO). We have previously shown that anti‐Id498 induces IgM anti‐A‐CHO secretion from B cells of donors that possess Id‐498+ antibodies in their serum. A‐CHO was presented to B cells either in soluble or insoluble form, i.e. coupled to beads or as intact bacteria. Purified blood B cell populations from three Id′ healthy donors with high numbers of circulating anti‐A‐CHO B cells were used and antibody‐producing B cells are enumerated in a single‐cell assay (spot ELISA). The data show that anti‐Id498 was superior in the induction of IgM anti‐A‐CHO‐secreting B cells in two donors (factor 4.6 and 13.5 as compared to the most efficient antigenic stimulation). In the third donor antigen stimulation was slightly more efficient than anti‐Id but only with Sepharose‐bound A‐CHO and not with soluble A‐CHO or intact bacteria. The increase of specific B cells induced after stimulation with anti‐Id498 could be abolished after addition of autologous T cells in two donors. On the contrary, an enhancement of the specific response was observed after addition of autologous T cells in antigen‐stimulated cultures. Neither suppression nor enhancement were induced by addition of irradiated T cells.
Regulatory role of the CD8 antigen in both CD3 and CD2 monoclonal antibody‐induced nonspecific cytotoxicity of class I‐ and class II‐allospecific cytotoxic T cell clones
Authors: Van Seventer Gijs A. , Van Lier Rene A. W. , Kuijpers Karel C. , Spits Hergen , Melief Cornelis J. M.
Published: March 2007

We investigated the function of the CD8 moiety in antigen‐specific and alternative activation of HLA class I‐ and HLA class II‐allospecific CD8+ cytotoxic T lymphocyte (CTL) clones, Monoclonal antibodies (mAb) directed against the CD8 structure were only found to inhibit antigen‐specific cytotoxicity of class I‐allospecific CD8+ CTL clones and not of a class II‐allospecific CD8+ CTL clone. However, cytotoxicity induced by CD3 mAb (used at suboptimal concentrations) or CD2 mAb in both types of CTL clone was blocked by CD8 mAb. The class II‐allospecific CD8+ CTL clone was uniformly more difficult to inhibit than the class I‐allospecific CD8′ CTL clones and, moreover, also easier to induce to exert nonspecific cytotoxicity by CD2 mAb and CD3 mAb. The absence of CD8 mAb blocking of antigen‐specific cytotoxicity of the class II‐specific CD8+ CTL clone is, therefore, assumed to result from too strong a triggering signal to be overcome by the down‐regulatory signal of the CD8 antigen. These combined findings suggest a down‐regulatory function of CD8 not only in T cell receptor (TcR)/CD3 activation, but also in TcR/CD3‐controlled alternative activation routes such as the CD2 activation pathway.
Clq acts synergistically with phorbol dibutyrate to activate CR1‐mediated phagocytosis by human mononuclear phagocytes
Authors: Bobak David A. , Frank Michael M. , Tenner Andrea J.
Published: March 2007

The adherence of human monocytes and culture‐derived macrophages to surfaces coated with complement subcomponent Clq has been previously shown to enhance Fc receptor (FcR)‐mediated phagocytosis by these cells. We examined the effects of Clq on C3b/C4b receptor (CR1)‐mediated phagocytosis by mononuclear phagocytes. A small percentage of human monocytes cultured in the presence of serum became competent to ingest sheep erythrocytes bearing IgM and C4b (EAC4b). This phagocytic activity was enhanced when these cultured‐derived macrophages were adhered to Clq‐coated surfaces. However, when cultured in a defined serum‐free medium, these cells did not ingest EAC4b, even in the presence of Clq. To investigate this differential responsiveness, we studied the effects of Clq in conjunction with cell‐activating agents on CR1 activation. Treatment of serum‐free cultured monocytes with phorbol dibutyrate (PDBu), prior to addition of the targets, induced these cells to ingest EAC4b. In addition, when exposed to Clq, both the percentage of these PDBu mononuclear phagocytes ingesting EAC4b and the number of targets ingested increased threefold over the level achieved by macrophages treated with PDBu alone. The chemoattractant N‐formyl‐methionyl‐leucyl‐phenylalanine did not activate CR1‐mediated phagocytosis and did not substitute for PDBu in causing synergy with Clq. Freshly isolated monocytes adhered to human serum albumin‐coated glass slides in the absence or presence of PDBu did not phagocytose EAC4b. Also Clq did not stimulate monocyte CRl‐mediated phagocytosis. However, addition of PDBu to cells adherent to the Clq surface triggered phagocytosis of EAC4b. The concentration of PDBu and the time of addition of PDBu relative to addition of the EAC4b targets were found to be important parameters for the achievement of maximal synergy in both the freshly isolated and cultured cell systems. This enhanced phagocytic activity was also seen with cells adhered to the purified collagen‐like, pepsin‐resistant, fragment of Clq. Since this region was previously shown to interact with Clq surface receptors, it appears that occupancy of this receptor is triggering events contributing to the enhanced cellular function. These experiments suggest that Clq and PDBu promote ingestion via CR1 by different but synergistic mechanisms. These data also demonstrate that the CR1‐mediated enhancement of phagocytosis is not specific for FcR‐mediated ingestion, but also applies to phagocytosis via CR1.
Characterization of the human type I interferon receptor by ligand blotting
Authors: Schwabe Michael , Princler Gerald L. , Faltynek Connie R.
Published: March 2007

The human type I interferon (IFN) receptor was characterized by ligand blotting. In this method, plasmalemma proteins or detergent‐lysed whole‐cell extracts from human Burkitt lymphoma cell lines were separated on polyacrylamide gels and subsequently transferred onto nitrocellulose sheets. Probing the blots with 3 × 10−10 M 125I‐labeled recombinant IFN‐αA (125I‐rIFN‐αA) revealed an IFN‐α‐binding protein with an apparent molecular mass of 95 kDa (p95). Performing the electrophoretic run under reducing conditions completely abrogated the signal on the blot, indicating that the type I IFN receptor contains a'disulfide bond essential for IFN binding. Optimal binding of 125I‐rIFN‐αA occurred at pH 9. The specificity of the binding reaction was established by simultaneously adding an excess of unlabeled IFN during incubation of the blots with 125I‐rIFN‐αA. The addition of either unlabeled IFN‐α or IFN‐β, but not IFN‐γ, abolished the binding of 125I‐rIFN‐αA to p95. 125I‐rIFN‐γ at 1.25 × 10−11 M bound to two proteins distinct from p95 with apparent molecular mass of 92 and 87 kDa, respectively. Saturability of 125I‐rIFN‐αA binding was demonstrated by probing a constant amount of membrane proteins with increasing amounts of 125I‐rIFN‐αA. Scatchard analysis of the binding data yielded an apparent Kd of 5.4 × 10−10 M for the immobilized type I IFN receptor. The expression of p95 on IFN‐α‐resistant and ‐sensitive cells was indistinguishable. We conclude that p95 is the IFN‐α/β receptor and that two proteins (p92 and p87) can specifically bind IFN‐γ. These results indicate that ligand blotting is a versatile method for characterization of unmodified IFN receptors and IFN‐receptor interaction and could also provide a new investigational approach for other cytokine receptor systems.
Orientation of epitopes influences the immunogenicity of synthetic peptide dimmers
Authors: Cox Josephine H. , Ivanyi Juraj , Young Douglas B. , Lamb Jonathan R. , Syred Andrew D. , Francis Michael J.
Published: March 2007

The immunogenicity of synthetic peptide dimers based on epitope sequences derived from the mycobacterial 65‐kDa antigen and the foot and mouth disease virus (FMDV) VP1 protein was examined in inbred mice. The analysis was directed towards the potential helper role of a T cell stimulatory mycobacterial epitope (65–85) with respect to poorly immunogenic sites either from the same molecule (422–436) or from VP1 (141–160). The 65–85 repeat homodimer induced an antibody response in CBA/ca but not in C57BL/6 mice, both nonresponders to the 65–85 monomer, and amplified the antibody response in BALB/c, monomer‐responder mice. Analysis of the immunogenicity of hybrid dimers in BALB/c mice showed that the orientation of peptides within the dimer is critical for the extent of the produced antibody response. Only the 422–436/65–85 but not the 65–85/422–436 induced antibodies binding to the 422–436 sequence which was nonimmunogenic when injected either as a monomer or dimer. Despite the striking difference in immunogenicity, both tested hybrid dimers reacted equally in the solid‐phase immunoassay with antisera raised to 65–85‐dimer or 422–436/65–85 peptides or with a monoclonal antibody to the 422–436 epitope. The described differences in antibody responsiveness also cannot be attributed merely to the extent of T cell stimulation since the proliferative responses were uniformly expressed for all relevant combinations of peptides. Antisera to 65–85 dimer and 422–436165–85 hybrid also reacted with the native 65‐kDa protein. Furthermore, the production of FMDV‐neutralizing antibodies in response to the 141–160 (VP1‐derived)/ 65–85 hybrid peptide in 141–160 nonresponder B10.D2 mice also confirmed the helper activity of the 65–85 epitope. Thus, combining heterologous peptides with the N‐terminal of the mycobacterial 65–85 sequence may be generally applicable for the potentiation of peptide vaccines.
Comparison of interleukin 2 and 12‐O‐tetradecanoyl‐phorbol 13‐acetate as signals for protein kinase C activation in purified human T lymphocytes
Authors: Mahé Yann , Piau Jean‐Pierre , Wakasugi Naomi , Tursz Thomas , Gacon Gerard , Wakasugi Hiro
Published: March 2007

Interleukin 2 (IL2) and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti‐CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes.

TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti‐CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti‐CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti‐CD3 mAb‐treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation.

In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high‐affinity IL2 receptors.
Phenotypical characterization of the cells invading pancreatic islets of diabetic BB/OK rats: effect of interleukin 2 receptor‐targeted immunotherapy
Authors: Hahn Hans J. , Gerdes Johannes , Lucke Sake , Liepe Larissa , Kauert Christiane , Voik Hans D. , Wacker Hans H. , Brocke Stephan , Stein Harald , Diamantstein Tibor
Published: March 2007

We investigated immunohistochemically the phenotypes of mononucleated cells invading pancreatic islets of diabetic BB/OK rats in comparison to the diabetesresistant parental strain, and 12 and 120 days after a temporary treatment (10 days) with a monoclonal antibody (1 mg/kg b.w.) directed against interleukin 2 receptor (IL 2R) combined with a subtherapeutic dose of cyclosporin A (1.5 mg/kg b.w.). Using a panel of monoclonal antibodies (OX‐19, OX‐8, W3125, KI‐M2R, OX‐6, OX‐17, ART‐18) and the alkaline phosphatase anti‐alkaline phosphatase system to visualize the bound primary antibodies, we observed an even distribution of mono‐nucleated cells across the endocrine pancreas at a “background” level when obtained from diabetes‐resistant parental rat strain. Diabetic BB/OK rats, characterized by a moderate hyperglycemia and a marked decrease of pancreatic insulin content, displayed a remarkable accumulation of mononucleated cells in the endocrine pancreas. Morphometric studies revealed an increase of all phenotypes investigated, nearly all mononucleated cells expressed class I1 histocompatibility antigens (OX‐6+, OX‐17+) and the number of cells expressing the IL 2R (ART‐18+) was markedly enhanced. Sixty‐seven percent of the immunotherapeutically treated BB/OK rats normalized plasma glucose and enhanced pancreatic insulin content. The successfully treated animals are characterized by a decrease of cells invading pancreatic islets (OX‐19+, OX‐8+, W3/25+, KI‐M2R+), a decrease of class II histocompatibility antigen and IL 2R expression. The number of IL 2R cells is also diminished in the endocrine pancreas of unsuccessfully treated BB rats.
Reversal by interferon‐γ of inhibition of delay ed‐type hypersensitivity induction by anti‐CD4 or antiinterleukin 2 receptor (CD25) monoclonal antibodies. Evidence for the physiological role of the CD4+ TH1+ subset in mice
Authors: Diamantstein Tibor , Eckert Rolf , Volk Hans‐Dieter , Kupier‐Weglinski Jerz‐W.
Published: March 2007

In mice, induction of delayed‐type hypersensitivity (DTH) and of antibody formation to sheep red blood cells (SRBC) was inhibited by the anti‐CD4 monoclonal antibody (mAb) GK1.5. However, anti‐interleukin 2 mAb AMT‐13 inhibited DTH response but not antibody formation. The inhibitory action of the mAb on DTH, but not an antibody formation, was reversed by concomitant injection of recombinant mouse interferon‐y. These data provide the first in vivo evidence for the postulated physiological role of the CD4+ TH1 lymphocyte subset and indicate that the action of anti‐interleukin 2 receptor mAb is restricted to this T helper subset.
Mechanism of skin allograft enhancement across an H‐2 class I mutant difference. Evidence for involvement of veto cells
Authors: Martin Kast W. , Twuyver Esther Van , Mooijaart Rob J. D. , Verveld Marieke , Kamphuis Alfons G. A. , Melief Cornelis J. M. , De Waal Leo P.
Published: March 2007

Intravenous injection of spleen cells across mutant class I H‐2 incompatibility results in a drastic donor‐specific prolongation of skin allograft survival and a marked decrease in the donor‐specific cytotoxic T lymphocyte precursor (CTLp) frequency. This immunosuppressive effect depends on the presence of radiosensitive T cells in the donor cell inoculum. It was excluded that a graft‐vs.‐host reaction was responsible for the observed effects. In mixing experiments, spleen cells from animals transfused with allogeneic lymphocytes could not suppress a normal CTL response against the alloantig, despite an excess of putative recipient‐derived spleen suppressor cells. The data are compatible with the idea that donor T cells function as veto cells which inactivate recipient CTLp directed against the alloantigen expressed by the veto cell.
Monoclonal antibodies which react with the T cell receptor γ/δ recognize different subsets of CD3WT31‐ T lymphocytes
Authors: Ferrini Silvano , Prigione Ignazia , Bottino Cristina , Ciccone Ermanno , Tambussi Giuseppe , Mammoliti Serafina , Moretta Lorenzo , Moretta Alessandro
Published: March 2007

A polyclonal CD34‐8‐WT31‐ cell line (termed SFG) was utilized for mice immunization in order to produce monoclonal antibodies (mAb) specific for the T cell receptor (TcR) γ/δ. Hybrid supernatants were screened for their ability to induce SFG cells (but not conventional TcR α/β CTL lines) to kill the murine Fc receptor‐positive P815 target cell line. Three hybrids, termed G1, A13 and F11, were isolated according to this screening. By indirect immunofluorescence G1 mAb reacted with 65% of SFG cells, while A13 stained 26% and F11 75% of cells. Double‐fluorescence analysis revealed that G1 and A13 mAb identify two distinct, non‐overlapping subsets of cells present in the SFG cell line. The reactivity of the mAb was also analyzed on a panel of representative TcR α/β clones. G1 mAb reacted with 5 clones, that were also stained by the previously described BB3 mAb (recognizing the disulfide‐linked form of TcRα/β). These clones failed to react with A13 and δ‐TCS‐1 mAb (the latter of which is known to react with a non‐disulfide‐linked form of TcR α/β). Out of six clones that reacted with A13 mAb, four were also δ‐TCS‐1, whereas two were δ‐TCS‐1‐ and none of them reacted with G1, (or BB3) mAb. In contrast to the mAb above, F11 brightly stained the G1A13‐ clones and more weakly the G1‐A13 clones. Moreover, F11 efficiently triggered both types of clones to kill the P815 target cells while G1 and A13 were able to trigger only G1 or A13 clones, respectively. None of the mAb above reacted with a large number of CD3WT31 clones. Antibodyinduced surface antigen modulation experiments indicated that molecules recognized by G1, A13 and F11 were physically associated on cell surface with CD3 determinants. In addition, immunoprecipitation followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis (performed on 125I‐surface‐labeled TcRα/β clones) revealed that molecules recognized by G1, A13 and F11 displayed an apparent mol. wt. corresponding to that of CD3‐associated TcR molecules, immunoprecipitated by anti‐CD3 mAb from the same clones.
T cell immunity or tolerance as a consequence of self antigen presentation
Authors: Lin Rong Hwa , Stockinger Brigitta
Published: March 2007

In this study we investigated the basis for immunity or tolerance to a mouse serum protein, the fifth component of complement (C5). In C5‐deficient mice this protein is absent from serum and therefore they are not tolerized. Immunization of C5‐deficient mice with C5‐sufficient serum generates CD4 T cells, which recognize C5 presented in the context of class II. No C5‐specific responses were observed in T cells from C5‐sufficient mice. We show that this self protein is processed and presented with class II by cells from C5‐sufficient tolerant mice and can be recognized by C5‐specific T cell clones and hybrids in the absence of exogenously added antigen. The stimulation of C5‐specific T cells by C5‐sufficient antigen‐presenting cells is not a consequence of C5 secretion and subsequent processing in vitro but rather employs C5 peptide/class II complexes generated in vivo. We conclude that this self antigen is presented in normal mice in a form recognizable by T cells to induce and maintain immunological tolerance.
Isolation of rat IgM to IgG hybridoma isotype switch variants and analysis of the efficiency of rat Ig in complement activation
Authors: Pluschke Gerd , Bordmann Gerard , Daoudaki Maria E. , Lambris John D. , Achtman Mark , Neibert Michael
Published: March 2007

Sequential sublining was used in combination with enzyme‐linked immunosorbent assays to isolate μ → γ isotype switch variants of the rat IgM secreting mouse‐rat B cell hybridoma line BA1.8. Switch variants to all four subclasses of IgG were obtained. The variant antibodies retained the antigen specificity of the parental IgM for the O18 (lipopolysaccharide) antigen of Escherichia coli. In sodium dodecyl sulfate‐polyacrylamide gels the apparent molecular mass of the γ heavy chains decreased in the order γ2b > γ1 > γ2c > γ2a.

IgM, IgG1, IgG2a, IgG2b and IgG2c of the BA1.8 variant family and IgG2b, IgE and IgA of the previously described BA1.2 family were used for a comparative analysis of the capacity of rat Ig to activate complement. Efficient lysis of sheep erythrocytes coated with the O18 antigen was observed with IgM and all IgG subclasses, but no lysis was triggered by IgE or IgA. One hundred to 1000 IgG molecules were required to mediate the same hemolytic activity as one IgM molecule. The four IgG subclasses were equally efficient at mediating lysis by rat or human complement, while IgG2a was less efficient with guinea pig complement than the other three IgG subclasses.

Antibody‐triggered binding of C3 to pathogenic O18: K1 E. coli bacteria was measured using serum containing 125I‐labeled C3. K1‐encapsulated strains did not fix C3 efficiently in the absence of specific antibodies while acapsular mutants fixed C3 via the alternative pathway. IgM and all IgG subclasses triggered C3 binding to the K1 encapsulated bacteria. The capacity of IgM to mediate C3 fixation was not greater than that observed with IgG.
A murine nephritogenic monoclonal anti‐DNA autoantibody binds directly to mouse laminin, the major non‐collagenous protein component of the glomerular basement membrane
Authors: Sabbaga Jorge , Line Sergio Roberto Peres , Potocnjak Pedro , Madaio Michael P.
Published: March 2007

The interaction of the murine monoclonal anti‐DNA antibody H241 with extracellular glomerular antigens was found to be due to the binding of this antibody to laminin, the major non‐collagenous protein constituent of the glomerular basement membrane. This interaction is specific, since it is inhibited by laminin, double‐stranded DNA and single‐stranded DNA in solution. Furthermore, the binding of H241 to mouse laminin is mediated by conformational properties of the antigen because mild denaturation of laminin strongly decreases the binding capacity of H241, while exposure of laminin to sodium dodecyl sulfate, completely abolishes this interation. H241 is able to bind to both, human and mouse laminin. These findings are in agreement with the ligand binding specificities of the autoantibodies spontaneously produced, that differ from those generated by artificial immunization. We conclude that the polyreactivity of H241 that confers to it the capacity to bind laminin, may account for its ability to form immune deposits by binding directly to non‐DNA glomerular antigens.
Intradomain H‐2 Kd/Dd recombinants define the same regions as crucial for recognition by alloreactive or major histocompatibility complex‐restricted cytolytic T cells
Authors: Maryanski Janet L. , Abastado Jean‐Pierre , Robson MacDonald H. , Kourilsky Philippe
Published: March 2007

To identify residues on class I major histocompatibility complex (MHC) antigens that are important in T cell recognition, we analyzed a series of 11 intradomain recombinant mouse MHC (H‐2) molecules in which N‐terminal H‐2Kd segments of varying lengths are followed by H‐2Dd segments. Lysis of L cell transfectant target cells by a series of alloreactive cytolytic T cell (CTL) clones specific for Kd or for Dd revealed several regions that contain residues critical for specific recognition. These residues map within the presumed antigen‐binding site of the MHC molecule. Of particular interest was the finding that the two regions identified as important for Kd allorecognition match those that influence the recognition of synthetic peptide antigens by Kd ‐restricted CTL.
Expression of the p75 interleukin 2‐binding protein on CD34‐8‐Tac‐ cells from autoimmune MRL/MP‐lpr/lpr mice
Authors: C. gutierrez‐Ramos José , Pezzi Luis , Palacios Ronald , Martínez‐A. Carlos
Published: March 2007

The recently described (Sharon, M. et al., Science 1986. 234: 859) interleukin 2 (IL 2)‐binding molecule p75 was detected in the CD34‐8‐Tac‐ “double‐negative” cell population selectively expanded in lupus‐like autoimmune mice MRL/MP‐lpr/lpr using cross‐linking studies. Scatchard analysis of the IL2 binding revealed the existence of approximately 4700 sites per cell with an apparent Kd of 1500 pM. The cell line LD1.T3B, derived from this population, shared surface markers and the p75 presence/p55 absence of IL 2‐binding proteins with its in vivo counterpart, displaying around 3100 sites per cell with a Kd of about 1300 pM. Functional studies showed that high doses of IL 2 had an inhibitory effect on the autonomous growth of this cell line in the absence of the development of killer activity. This study provides evidence of the functional abilities of p75, and shows that the use of Tac/p55 surface expression only to evaluate IL 2 receptors and T cell activation can be an oversimplification as well as misleading.
Human T cell clones with specificity for insulinoma cell antigens
Authors: Vliet Els Van , Roep Bart O. , Meulenbroek Lex , Jan Bruining G. , De Vries René R. P.
Published: March 2007

Several lines of evidence suggest that islet‐specific T cells are important in the pathogenesis of the insulitis resulting in insulin‐dependent diabetes mellitus (IDDM). Therefore, we decided to analyze islet‐specific T cell reactivity in the peripheral blood of IDDM patients. With the use of insulinoma membranes as antigen, T cell lines were generated from peripheral blood mononuclear cells of patients with recent onset of the disease. In a proliferation assay such T cell lines responded to insulinoma membranes and, though to a lesser extent, also to fibroblast membranes, the control antigen used. One of the T cell lines was cloned. Eight clones were isolated that respond to insulinoma antigens. Five of these eight clones appeared to be specific for insulinoma membranes, i.e. they demonstrated proliferation in response to insulinoma but not fibroblast membranes. These insulinoma‐specific proliferative responses are HLA‐DR restricted.
Differential activation of cytokine genes in normal CD4‐bearing T cells is stimulus dependent
Authors: Carding Simon R. , West Jeffrey , Woods Andrea , Bottomly Kim
Published: March 2007

Studies of cloned CD4 T cell lines have shown that they can be separated into two distinct subsets with distinctions in their functional capabilities and by the differential release of either interleukin 2 (IL 2) (TH1/inflammatory type) or IL 4 (TH2/helper type) upon activation. To establish if in vivo‐derived CD4 T cells can exhibit distinct subsets we have investigated whether normal CD4 T cells demonstrate differential expression of IL 2 and IL 4 mRNA, and secretion of IL 2 and IL 4 after primary stimulation in vitro.

Utilizing the technique of in situ hybridization IL 2 and IL 4 gene expression in individual CD4 T cells was readily detectable after concanavalin A (Con A) phytohemagglutinin (PHA) or pokeweed mitogen (PWM)‐mediated activation. The frequencies of activated T cells producing IL 2 and IL 4 mRNA after Con A or PHA activation were approximately equivalent (30–40% of cells); however, after PWM activation the number of CD4 T cells expressing IL 4 mRNA (78%) was more than twofold greater than the number of cells producing IL 2 mRNA (30%). Maximal levels of IL 2 gene expression occurred 24 h after mitogen activation whereas the highest levels of IL 4 mRNA were not detected until 48 h after mitogen activation. Similar distinctions in the kinetics of IL 2 and IL 4 secretion after mitogen activation were also found demonstrating good concordance in the observed expression of IL 2 and IL 4 mRNA and the levels of secreted lymphokines detected by bioassay. Most importantly, we have shown by in situ hybridization analysis that the majority of individual CD4 T cells produce only IL 2 or IL 4 mRNA, and not both, after primary activation in vitro. By contrast, most CD4 T cells activated in the presence of PMA and ionophore express both IL 2 and IL 4 mRNA.

Our studies demonstrate that in normal, non‐clonal populations of CD4 T cells, the production of IL 2 and IL 4 is independently regulated in the majority of cells and appears to be stimulus dependent.
Cytotoxic T lymphocytes in reticuloendotheliosis virus‐infected chickens
Authors: Weinstock Daniel , Schat Karel A. , Calnek Bruce W.
Published: March 2007

Cytotoxic T lymphocytes were functionally demonstrated in spleen cells from chickens 7 days post inoculation with reticuloendotheliosis virus using a Cr‐release assay. Major histocompatibility complex (MHC)‐restricted cytotoxicity was demonstrated using effector and target cells from two different strains of chickens of known avian MHC haplotype. Anti‐viral specificity was shown and in vivo generation of MHC‐restricted cytotoxicity was evaluated. Cytotoxic T cells were distinguished from macrophages and natural killer cells. Their cytotoxicity was not antibody dependent. Higher levels of cytolysis were found with cytotoxic T cells from embryonally bursectomized vs. intact chickens over a large range of effector to target cell ratios. Using monoclonal antibodies, cytotoxic T cells were further defined as Ia T cells by immunofluorescence, antibody plus complement‐mediated lysis of effector cells and blocking of cytolysis in the Cr‐release assay.
T cell receptor β chain usage in myelin basic protein‐specific rat T lymphocytes
Authors: Chluba Johanna , Steeg Christiane , Becker Andrea , Wekerle Hartmut , Epplen JöRg T.
Published: March 2007

Myelin basic protein (MBP)‐specific T cell lines and clones have been established from rats of the major histocompatibility complex (MHC)‐compatible Lewis and BS strains. All lines and clones are MHC class II restricted and share the CD4 phenotype. The cells proliferate specifically in response to either a peptide representing amino acids #68–88 of guinea pig MBP, to residues #47–67 or to an unidentified myelin antigen which is distinct from MBP. All lines and clones specific for MBP express the same T cell receptor (TcR) variable (V) β chain element, which is homologous to the mouse Vβ8.2 gene segment. Three lines/clones with the same antigen fine specificity have identical VβDβJβ junctions on the protein level, a region which represents part of the potential antigen‐binding protion of the TcR; two of the lines express members of the Vα2 family. These results suggest biased usage of TcR Vβ elements in rat T cells specific for MBP. Our findings broaden the basis for a rational therapeutic strategy to specifically intervene in the rodent model system of experimental allergic encephalomyelitis.
L3T4 T cells promoting susceptibility to murine cutaneous leishmaniasis express the surface marker Ly‐24 (Pgp‐1)
Authors: Moll Heidrun , Scollay Roland
Published: March 2007

In a murine model of cutaneous leishmaniasis, the importance of T cell‐dependent immunity has been documented by the susceptibility to parasite infection of athymic nude mice of both genetically resistant and genetically susceptible strains. T lymphocytes from uninfected mice have the capacity to promote resistance to Leishmania major infection in nude recipients, whereas T cells from mice chronically infected with L. major not only fail to mediate protection, but totally abrogate the host‐protective effect of normal mouse lymphocytes. Both these effects are mediated by T cells which have the phenotype L3T4Ly‐2‐. To discriminate between the two activities, we have tried to separate the L3T4 population on the basis of additional cell surface markers and we have found that peripheral L3T4 lymphocytes could be subdivided according to their expression of the Ly‐24 (Pgp‐1) surface marker. In adoptive transfer experiments, both the Ly‐24 and the Ly‐24 subset of L3T4 cells from uninfected mice had the capacity to mediate resistance to infection with L. major. However, diseasepromoting activity was only found in the L3T4Ly‐24 and not in the L3T4Ly‐24‐ subset of cells from mice with chronic cutaneous disease. Moreover, Ly‐24 expression was strongly increased in lymphocytes from chronically infected mice and in vitro limiting dilution analysis confirmed that the vast majority of L. major‐reactive T cells was L3T4Ly‐24. In genetically susceptible mice with chronic cutaneous leishmaniasis, Ly‐24 therefore appears to be a marker for lymphocytes with the capacity to abrogate resistance to disease, these cells being activated and expanding in the course of progressive L. major infection. Ly‐24 expression is a useful tool for phenotypic identification and selective enrichment of antigen‐activated and possibly memory T cells. It may facilitate the isolation of L. major‐specific T cell clones with defined activities.
The autoantigenic response to rabbit cytochrome c
Authors: Cooper Helen M. , Klinman Norman R. , Paterson Yvonne
Published: March 2007

In this report we describe the production and characterization of autoantibody responses to rabbit cytochrome c (cyt c) in rabbits immunized with either the native monomeric or polymerized form of rabbit cyt c. Fine‐specificity analyses of the response indicated that the majority of the response was to the evolutionarily conserved ammo‐terminal region of the molecule. The relative affinity of the autoantibody interaction with rabbit cyt c was assessed by a solid‐phase assay and was found to be lower than that observed for rabbit anti‐horse cyt c antibody populations. These findings are consistent with the prediction that low‐affinity self‐reactive B cells may escape tolerance induction.
MLR3 molecule is an activation antigen shared by human B, T lymphocytes and T cell precursors
Authors: Risso Angela , Cosulich Maria Elisabetta , Rubartelli Anna , Mazza Maria Rosaria , Bargellesi Antonio
Published: March 2007

MLR3 molecule is a membrane glycoprotein (mol. mass range 28–34 kDa) present on activated, but not resting human peripheral T cells, B cells and thymocytes. Its kinetics of appearance on the cell surface (3 h after the addition of the inductive signal to the cells) suggests that it is an early activation antigen. The proliferative response of cultured T and B lymphocytes and thymocytes to different activation signals is inhibited by the addition of MLR3 monoclonal antibody. Moreover the antibody in combination with non‐mitogenic doses of phorbol myristate acetate leads to proliferation of thymocytes and resting B and T lymphocytes. In the latter, synthesis of interleukin 2 is also induced. Biochemical analysis of MLR3 antigen indicates that it is a phosphorylated protein with N‐linked sugar moieties. Together these data suggest a role for MLR3 antigen in the signal transduction process during activation, both for mature lymphocytes and for T cell precursors.
The gene for human lymphocyte homing receptor is located on chromosome 11
Authors: Forsberg Ulla HÉLena , Ala‐Kapee Minna Maria , Jalkanen Sirpa , Andersson Leif C. , Schröder Jim
Published: March 2007

The mouse monoclonal antibody Hermes‐3 recognizes the human lymphocyte homing receptor. A panel of mouse‐human T lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, was analyzed for expression of human homing receptor by indirect immunofluorescence and immuno‐precipitation of radiolabeled cell lysates with Hermes‐3 antibody. Karyotypic analysis of the tested clones showed that the expression of human homing receptor correlated to the presence of human chromosome 11 in all but one clone. However, con‐canavalin A induced a weak to moderate expression of the homing receptor in this clone, but not in a chromosome 11‐ clone. Another clone, heterogeneous for the expression of homing receptor, was separated into a Hermes‐3 and a Hermes‐3‐ fraction with a fluorescence‐activated cell sorter. Karyotypic analysis performed after sorting showed human chromosome 11 to segregate with the Hermes‐3 antigen. To confirm these data we correlated the expression of two chromosome 11‐coded antigens, Trop‐4 and Leu‐7, with the expression of the homing receptor. In our hybrid clones these three antigens were expressed concordantly. The gene coding for the human lymphocyte homing receptor recognized by Hermes‐3 is thus assigned to chromosome 11.
Anti‐viral protection and prevention of lymphocytic choriomeningitis or of the local footpad swelling reaction in mice by immunization with vaccinia‐recombinant virus expressing LCMV‐WE nucleoprotein or glycoprotein
Authors: Hany Manuela , Oehen Stephan , Schulz Manfred , Hengartner Hans , Mackett Michael , Bishop David H. L. , Overton Hilary , Zinkernagel Rolf M.
Published: March 2007

The viral antigen specificity of primary cytotoxic T cell responses (CTL) of H‐2b, H‐2k, H‐2q, H‐2s, H‐2f and some H‐2‐recombinant mice against lymphocytic choriomeningitis virus (LCMV‐WE isolate) as well as the specificity of some CTL clones and T cell lines was defined on target cells infected with vaccinia‐recombinant virus expressing nucleoprotein (Np) or glycoprotein (Gp). Np was recognized together with H‐2q (Dq), H‐2d (DLd), H‐2s and H‐2b (Db). Gp specificity was restricted to H‐2f and H‐2b (Kb and Db); H‐2k‐restricted CTL anti‐LCMV responses were neither Gp nor Np specific.

The anti‐viral protective immunity induced by vaccinia‐Gp or vaccinia‐Np recombinants was evaluated in mice. In vivo protection was T cell mediated by class I restricted Ly‐2 T cells; it correlated well with the CTL specificity defined in vitro. Some of the CTL‐nonresponder H‐2 allele plus Np or H‐2 plus Gp combinations were, however, protected to variable and low degrees by vaccinia‐recombinant viruses, indicating that anti‐viral protection is a more sensitive readout for CTL activity than the in vitro assay. For example, B10.D2 H‐2d mice generated measurable CTL responses only to Np; after immunization with a vaccinia‐Np recombinant, LCMV titers were 104 times lower in spleens than in vaccinia‐primed controls. Although vaccinia‐Gp‐immunized BALB/c mice revealed no CTL activity in vitro, they nevertheless had 102 times lower LCMV titers in spleens than controls. Anti‐viral protection, particularly in low‐responder combinations, was usually short‐lived and diminished after 3 weeks. In a high‐responder situation, protection was of a longer duration (> 8 weeks). Vaccination with vaccinia‐Np or Gp recombinants protected mice against lethal T cell‐mediated lymphocytic choriomeningitis induced by LCMV or prevented the local footpad swelling reaction; these in vivo effects were H‐2 dependent and followed the identical roles established for CTL recognition in vitro.
Serological detection and molecular localization of allelic HLA‐DP supertypic epitopes
Authors: Colonna Marco , Tanigaki Nobuyuki , Tosi Roberto , Ferrara Giovan Battista
Published: March 2007

A DP serological allospecificity was identified using 125I‐labeled preparations of HLA class II molecules isolated from cells of HLA homozygous typing cell lines, SLE (DRw6, DQw1, DPw3) and WT46 (DRw6, DQw1, DPw2), and depleted of DR molecules by absorption with an anti‐DR monoclonal antibody. The specificity, provisionally called WT, was carried by class II molecules possessing the characteristics of DP molecules on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and detected primarily in DPw1, DPw3 and DPw5 cells and exceptionally in some DPw2 cells including WT46 and DPw4 cells on a large panel of DP‐pretyped B cell lines mostly derived from the 10th International Histocompatibility Workshop B cell reference panel. It was apparently allelic to another DP serological specificity BUT previously defined on DP molecules isolated from cells of DPw2 HLA deletion mutant cell line LCL 721.82. On the same cell panel, the BUT specificity was negative in all DPw1, DPw3 and DPw5 cells, and positive in all DPw2 and DPw4 cells and also in DPw2B and DPw4B cells except the cells typed WT. This DP association pattern was similar to that of the known allelic sequences, GGPM and DEAV, in DP ß F segment, one of the six variable segments in the second exon of DP ß gene. Thus, genomic DNA from 22 B cell lines pretyped for BUT and WT specificities were enzymatically amplified for the second exon of DP ß gene by the use of locus‐specific oligonucleotide primers and hybridized with 32P‐labeled oligonucleotide probes corresponding to the F segment sequence variations. This oligonucleotide typing showed perfect one‐to‐one correlation with the BUT and WT serological typing. The typing also revealed that WT46 cells, although typed DPw2, have the DEAV sequence common to DPw1, DPw3 and DPw5.
Feedback regulation of murine Ly‐1 B cell development
Authors: Lalor Paul A. , Herzenberg Leonore A. , Adams Sharon , Stall Alan M.
Published: March 2007

Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly‐1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19: 501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly‐1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly‐1 B cells reappear shortly after the treatment antibody disappears. This recovery, we also show, is prevented by restoring allotype‐congenic Ly‐1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype‐congenic peritoneal cells, sorted Ly‐1 B cells or a monoclonal population of Ly‐1 B “tumor” cells.

These findings in essence reveal a feedback mechanism through which mature Ly‐1 B cells prevent further Ly‐1 B cell development from Ig‐ precursors. This feedback regulation is independent of Ig secretion by the mature Ly‐1 B cells, since the monoclonal Ly‐1 B “tumor” population that prevents endogenous Ly‐1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly‐1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly‐1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly‐1 B population, which survives through self‐replenishment in adults, in accord with conditions that influence Ly‐1 B development during neonatal life.
The majority of mouse spontaneous rosette‐forming cells of thymic origin belong to the Ly‐2+ subset
Authors: Lepault Françoise , Fache Marie‐Pierre , Gagnerault Marie‐Claude , Bach Jean‐François , Dardenne Mireille
Published: March 2007

Spontaneous sheep erythrocyte rosette‐forming T cells (T‐sRFC) found in normal mouse spleen are specific for the xenogeneic species of red cells and are distinct from human E rosettes as illustrated by their persistence after sheep red blood cell (SRBC) incubation with anti‐LFA‐3 monoclonal antibodies directed against SRBC molecule binding to the E rosette receptor. We confirm here using monoclonal antibodies that T‐sRFC are Thy‐1+CD3+. Additionally, using cell separation techniques based on panning and cell sorting, it is shown that the vast majority of spleen sRFC have the L3T4−Ly‐2+ phenotype. As already shown for anti‐Thy‐1 antibodies, anti‐CD3 and anti‐Ly‐2 antibodies block rosette formation, whereas antibodies to more abundant cell surface antigens (T200, H‐2Db) are not inhibitory. These data suggest that the interaciton of T‐sRFC with SRBC occurs at or near the T cell receptor.
A monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different cytokines
Authors: Bottomly Kim , Luqman Mohammad , Greenbaum Laurence , Carding Simon , West Jeff , Pasqualini Theresa , Murphy Donal B.
Published: March 2007

CD4 T cell clones have been shown to be functionally heterogeneous in the mouse. However, it is not known if normal CD4 T cells are also functionally heterogeneous, or whether functional specialization is a result of cloning and long‐term culture. To approach this question, a monoclonal antibody reacting with a subset of CD4 T cells has been prepared by immunization of rats with different cloned T cell lines all sharing the same functional activity. This monoclonal antibody reacts with a subset of CD45 (T200) molecules by binding to a determinant requiring the expression of the second variable exon of the CD45 molecule. Some CD4 T cells bear high levels of this marker, while others react only weakly. This antibody was used to separate CD4 T cells into two subpopulations. The brightly staining population was found to produce interleukin (IL) 2 and not IL 4, while the weakly staining population produced IL 4 and not IL 2. These data demonstrate that CD 4 T cells in normal mice are already functionally committed, and that they differentially express forms of CD45 that contain the second variable exon.
A monoclonal antibody to LFA‐3, the CD2 ligand, specifically immobilizes major histocompatibility complex proteins
Authors: Bierer Barbara E. , Golan David E. , Brown Carl S. , Herrmann Steven H. , Burakoff Steven J.
Published: March 2007

T cells are activated when the antigen‐specific T cell receptor recognizes antigen in association with major histocompatibility complex (MHC) proteins. The T cell surface protein CD2 (T11, LFA‐2, the T erythrocyte receptor) and its target or stimulator cell ligand, lymphocyte function‐associated antigen‐3 (LFA‐3), are also involved in T cell adhesion and activation. The molecular mechanisms by which the CD2/LFA‐3 interaction affects T cell adhesion and activation are unclear. The CD2/LFA‐3 interaction may be modeled by the interaction between LFA‐3 and anti‐LFA‐3 monoclonal antibody (mAb). We used the fluorescence photobleaching recovery technique to investigate the effect of anti‐LFA‐3 mAb on the lateral mobility of MHC proteins in plasma membranes of JY, a human Epstein‐Barr virus‐transformed B cell line. Anti‐LFA‐3 mAb induced immobilization of class I MHC proteins labeled with bivalent but not monovalent fluorescein‐conjugated W6/32 mAb. Anti‐LFA‐3 mAb also caused immobilization of class II MHC proteins labeled with bivalent fluoresceinated LB3.1 mAb. In contrast, anti‐LFA‐3 mAb did not affect the mobilities of either a B cell membrane protein labeled with bivalent fluoresceinated anti‐CD45 (human leukocyte antigen) mAb or a membrane lipid analogue. Unlike anti‐LFA‐3 mAb, anti‐LFA‐1 mAb did not affect class I MHC protein mobility. These results suggest that CD2 binding to LFA‐3 may trigger a physiological response in which target cell MHC proteins, cross‐linked by receptors on the T cell surface, are immobilized at and thereby localized to the T cell‐target cell interface.