bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐10 inhibits the primary allogeneic T cell response to human epidermal Langerhans cells
Authors: Péguet‐Navarro Josette , Moulon Corinne , Caux Christophe , Dalbiez‐Gauthier Catherine , Banchereau Jacques , Schmitt Daniel
Published: March 2007

In this study, we analyzed the effect of interleukin‐10 (IL‐10) on the primary allogeneic T cell response induced by human Langerhans cells (LC), the dendritic cells from epidermis. We showed that IL‐10 strongly inhibited the T cell response, provided it was added at the beginning of the mixed epidermal cell lymphocyte reaction (MELR). Proliferation of both CD4+ and CD8+ T cell subsets was affected by the cytokine. An inhibitory effect of IL‐10 on human LC allostimulatory function was evidenced by the fact that IL‐10‐preincubated LC, but not IL‐10‐preincubated T cells, can display inhibitory effect. LC treatment with IL‐10 partially inhibited the increase of HLA‐DR expression on cultured LC as the percentage of highly positive HLA‐DR cells was lower than that observed in the absence of the cytokine. IL‐10 inhibited T cell alloreaction induced by 2‐day‐cultured human LC which constitutively display high levels of HLA class II, as well as ICAM‐1 and LFA‐3 antigens. This suggests that the suppressive effect of the cytokine was not merely related to an impaired up‐regulation of these molecules. Addition of IL‐1 during the MELR potentiated the allogeneic T cell proliferation and could reverse, at least partly, the inhibitory effect of IL‐10. Collectively, these data indicate that IL‐10 can prevent the alloreaction induced by human dendritic cells, providing new insights into the potential clinical use of this cytokine.
Isolation of tumor cell‐specific single‐chain Fv from immunized mice using phage‐antibody libraries and the re‐construction of whole antibodies from these antibody fragments
Authors: Kettleborough Catherine A. , Ansell Keith H. , Allen Richard W. , Rosell‐Vives Elisabet , Güssow Detlef H. , Bendig Mary M.
Published: March 2007

Enhanced expression of epidermal growth factor receptor (EGFR) occurs on a variety of malignant tissues thus making anti‐EGFR antibodies possible agents for the diagnosis and therapy of human tumors. Standard hybridoma technology has been used successfully to isolate anti‐EGFR antibodies from immunized mice and rats. This report demonstrates that phage‐antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mice. Anti‐EGFR antibodies were isolated from phage‐antibody libraries constructed not only from the spleen of an immunized mouse but also from the draining lymph node of an immunized mouse and from in vitro immunized mouse cells. Two of the single‐chain Fv isolated from the phage‐antibody libraries were engineered to create partially humanized whole antibody molecules.
Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells
Authors: Taylor‐Robinson Andrew W. , Liew Foo Y. , Severn Alison , Xu Damo , McSorley Stephen J. , Garside Paul , Padron Julio , Phillips R. Stephen
Published: March 2007

The balance between T helper type 1 (Th1) and T helper type 2 (Th2) cells determines the outcome of many important diseases. Using cloned murine T cell lines, evidence is provided that Th 1, but not Th 2, cells can be activated by specific antigens or a T cell mitogen, concanavalin A, to produce large amounts of nitric oxide (NO). Furthermore, NO can inhibit the secretion of interleukin (IL)‐2 and interferon‐γ by Th 1 cells but has no effect on IL‐4 production by Th 2 cells. Th 1 and Th 2 cells can, thus, be distinguished by their differential production of and susceptibility to NO. NO exerts a self‐regulatory effect on Th 1 cells which are implicated in immunopathology.
Defective T cell receptor/CD3 complex signaling in human type I diabetes
Authors: Maria Ruggero De , Todaro Matilde , Stassi Giorgio , Blasi Francesco Di , Giordano Marco , Galluzzo Aldo , Giordano Carla
Published: March 2007

The autoimmune process leading to the destruction of pancreatic β‐cells is mediated by T lymphocytes. Peripheral T cells from subjects with preclinical and clinical type I diabetes respond weakly in vitro to lectin stimulation. We, therefore, investigated in a group of newly diagnosed diabetic patients the presence of a defect in the signal transduction pathway of the T cell receptor (TcR)/CD3 complex.

Following stimulation with anti‐CD3‐coupled beads, the proliferative response in diabetic T cells was significantly decreased in comparison with that from normal T cells. Interestingly, addition of either recombinant interleukin (IL)‐2 or phorbol 12‐myristate 13‐acetate to the cell culture was able to completely restore impaired anti‐CD3‐induced proliferation in diabetic T cells, suggesting the presence of a defect through the TcR/CD3 pathway, located upstream of protein kinase C (PKC) activation and resulting in low IL‐2 production and proliferation. Intracellular Ca2+ measurements by Fluo‐3 labeling and flow cytometry analysis on diabetic and control T cells after anti‐CD3 stimulation gave comparable results, indicating that this defect does not involve events leading to intracellular Ca2+ mobilization. In contrast, anti‐CD3 stimulation of diabetic T cells resulted in a marked impairment of PKC translocation and CD69 antigen expression, as assessed by peptide substrate phosphorylation and by flow cytometry analysis, respectively. Taken together, our data clearly show the presence in individuals at the onset of the disease of an in vitro defect in the signal transduction pathway of the TcR/CD3 complex, resulting in ineffective PKC activation which is not able to induce normal IL‐2 production and proliferation of diabetic T cells.
Adhesion molecules utilized in binding of intraepithelial lymphocytes to human enterocytes
Authors: Kelleher Dermot , Murphy Anne , Lynch Sara , O'Farrelly Cliona
Published: March 2007

The expression of adhesion molecules by human duodenal intraepithelial lymphocytes (IEL) was examined by two‐color flow cytometry. Resting IEL expressed LFA‐1, HML‐1, CD44. Stimulation with phytohemagglutinin (PHA) resulted in down‐regulation of expression of these molecules with induction of expression of ICAM‐1 and VLA‐4. VLA‐4 expression was also found on non‐activated IEL from patients with celiac disease. In addition, IEL expressed an antigen recognized by a novel monoclonal antibody D2.1. The molecular mass of D2.1 is heterogenous: 82 kDa in peripheral blood lymphocytes and 44 kDa in an IEL line. Expression of this antigen was also up‐regulated by PHA. To determine the involvement of these antigens in binding of IEL to human enterocytes, we developed a system based on adherence of an IEL cell line to the I407 fetal intestinal cell line. Monoclonal antibodies VLA‐4, D2.1 and to a lesser extent ICAM‐1 blocked adherence of IEL to I407 cells. These data suggest that VLA‐4 and D2.1 may be involved in adherence of IEL to human enterocytes or secreted matrix molecules in vivo.
Isolation of peritoneal precursors of B‐1 cells in the adult mouse
Authors: Marcos Miguel A. R. , Gaspar María L. , Malenchere Evelyne , Coutinho Antonio
Published: March 2007

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B‐1 cells, followed by the appearance of a population of B220+IgM−lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre‐B cells expression of λ5, VpreB, and RAG‐1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220+IgM− cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B‐1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220+IgM− cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre‐B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM‐plasma cell/B cell numbers in the spleens of peritoneal pre‐B cell recipients is more than 500‐fold higher than that of recipients reconstituted by BM pre‐B cells. We take these data to indicate that (1) differentiative commitment to the B‐1 cell population occurs before selection events on mature cells; (2) B‐1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage‐related differential ability of mature B cells to undergo terminal differentiation to high‐rate Ig secretion.
Dissection of T cell antigen receptor signaling using protein tyrosine kinase inhibitors
Authors: Baldari Cosima T. , Telford John L.
Published: March 2007

T cell antigen receptor (TcR) recognition of appropriately presented antigen results in the rapid activation of protein tyrosine kinases. Subsequent events include activation of protein kinase C and increased intracellular free calcium which lead to the activation of transcription factors involved in regulating interleukin‐2 gene expression. We have assayed the ability of a panel of protein tyrosine kinase (PTK) inhibitors to interfere with activation of the NF‐AT transcription factor by TcR ligation or treatment with phorbol ester and calcium ionophore which bypass many of the early events of TcR signal transduction. The results indicate that PTK are involved in early and late stages of TcR signaling. Moreover, one inhibitor (genistein) revealed the existence of a PTK which down‐regulates specifically calcium‐mediated signaling at a point downstream of the PTK p56lck but upstream of calcium mobilization.
Analysis of susceptibility of mature human T lymphocytes to dexamethasone‐induced apoptosis
Authors: Tuosto Loretta , Cundari Enrico , Montani Maria Saveria Gilardini , Piccolella Enza
Published: March 2007

We present evidence that dexamethasone (Dex), a synthetic glucocorticosteroid, causes apoptosis in mature human T cells, similarly to what has been reported for murine T lymphocytes. Human T cell clones and short‐term activated T lymphocytes treated with Dex show the characteristic pattern of apoptotic cells, such as hypodiploid nuclei, chromatin condensation and DNA fragmentation into oligonucleosomal fragments. However, Dex susceptibility of T cells to apoptosis is cell cycle‐dependent. The progression in the proliferative cell cycle (G1 versus S) rescues Dex‐treated T cells from apoptosis. Moreover, occupancy of the T cell receptor reverses Dex‐induced apoptotic phenomena. These observations suggest that glucocorticoids contribute to the regulation of the proliferative or the suicidal response of antigen‐activated human T cells.
Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity
Authors: Mouritsen Søren , Meldal Morten , Christiansen‐Brams Ida , Elsner Henrik , Werdelin Ole
Published: March 2007

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81–96)‐Y (here named 1) which is immunogenic in H‐2k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule Ek, was synthesized in five different glycosylated forms. The N‐terminal serine of HEL(81–96)‐Y was derivatized with D‐glucose (2), maltotriose (3), and a branched D‐glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D‐glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non‐glycosylated peptide, labeled with 125I, to bind to the two MHC class II molecules, Ak and Ek, was studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to Ek. Surprisingly 2, 3 and 4 bound better to Ek than did the non‐glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to Ek of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non‐glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant α‐helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non‐α‐helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.
Degeneracy of T cell receptor recognition of an influenza virus hemagglutinin epitope restricted by HLA‐DQ and ‐DR class II molecules
Authors: Jones Claerwen M. , Lake Richard A. , Lamb Jonathan R. , Faith Alex
Published: March 2007

The T cell receptor (TcR) recognizes antigens in the form of short peptide fragments bound to major histocompatibility (MHC) molecules. TcR have an immunoglobulin (Ig)‐like structure and, in an analogous manner to antigen recognition by Ig, the third complementarity determining regions (CDR3) of the TcR are believed to provide the primary contact with the peptide lying in the MHC groove. CDR1 and CDR2 are thought to contact the presenting MHC molecule. We have analyzed seven human CD4+ T cell clones that recognize a conserved peptide epitope (residues 255–270) within the influenza virus hemagglutinin (H3) HA1 subunit. Two T cell clones recognized the peptide in the context of HLA‐DRB1*1001 and HLA‐DQB1* 0602/DQA1*0102, respectively, and shared Vα, Vβ and Jβ gene segments. Only the junctional regions encoding the CDR3 regions of the two TcR chains were different. This suggests that the CDR3 regions of these TcR interact with the MHC class II molecule. Six of the T cell clones were restricted by the HLA‐DRB1*1001. Two of these T cell clones expressed Vβ9.1 and three expressed Vβ13 gene segments; the remaining clone expressed Vβ7.2, a close homologue of Vβ9.1. A diverse selection of Vα and J gene segments contributed to the junctional heterogeneity of the TcR, indicating a diversity of sequence combinations recognizing the epitope. Nevertheless, five out of six T cell clones bore a motif in the Vα CDR3 loop consisting of adjacent acidic and polar amino acid residues, eight residues from the carboxyl end of each CDR3.
Essential role for natural killer cells in the lethal lipopolysaccharide‐induced Shwartzman‐like reaction in mice
Authors: Heremans Hubertine , Dillen Chris , van Damme Jo , Billiau Alfons
Published: March 2007

Observations in our laboratory have provided evidence that interferon‐γ (IFN‐γ) is a key regulator of inflammatory responses to bacterial lipopolysaccharide (LPS) (Heremans et al., J. Exp. Med. 1990. 171: 1853): treatment of mice with neutralizing monoclonal antibody against IFN‐γ was found to completely prevent lethal shock reactions, in particular the generalized Shwartzman reaction, whereas treatment with IFN‐γ sensitized the mice to the development of such reactions. Since activated T cells and natural killer (NK) cells are the main if not the only potential source of LPS‐induced IFN‐γ, we investigated the relative importance of these cells in the development of the generalized Shwartzman‐like reaction in mice by depleting them selectively with relevant monoclonal antibodies. Treatment with antibodies directed against the CD4+ T cell subset was not effective in protecting mice. Anti‐CD8 antibody did attenuate the reaction to some extent. However, markedly reduced mortality was seen in mice which were depleted of NK cells by systemic administration of polyclonal anti‐asialo GM1 or monoclonal anti‐NK1. 1 antibodies. Failure of T cells to promote the Shwartzman reaction was also evidenced by the observation that thymus‐less nude mice, which are deficient in T cells, were more rather than less sensitive to the reaction. Approximately 20 times less LPS was needed to induce the lethal reaction in these mice than in NMRI mice and 58 times more anti‐IFN‐γ antibody was required to block mortality. Nu/nu mice reportedly have an over‐active NK cell compartiment. IFN‐γ production by these cells in LPS‐treated mice may account for the augmented sensitivity. Our data suggest that NK cells may be the most important source of endogenous IFN‐γ which mediates the LPS‐induced lethal reactions in mice.
Interleukin‐10 controls interferon‐γ and tumor necrosis factor production during experimental endotoxemia
Authors: Marchant Arnaud , Bruyns Catherine , Vandenabeele Peter , Ducarme Martine , Gérard Catherine , Delvaux Anne , De Groote Donat , Abramowicz Daniel , Velu Thierry , Goldman Michel
Published: March 2007

Interleukin‐10 (IL‐10) is a potent inhibitor of lipopolysaccharide (LPS)‐induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN‐γ is another important mediator of LPS toxicity, we studied the effects of IL‐10 on LPS‐induced IFN‐γ synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL‐10 (rhIL‐10) (10 U/ml) to human whole blood markedly suppressed LPS‐induced IFN‐γ release while neutralization of endogenously synthesized IL‐10 resulted in increased IFN‐γ levels. The ability of rIL‐10 to inhibit LPS‐induced IFN‐γ synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL‐10 (rmIL‐10) 30 min before and 3 h after challenge of BALB/c mice with 100 μg LPS resulted in a threefold decrease in peak IFN‐γ serum levels. We then examined the production and the role of IL‐10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL‐10, peak IL‐10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL‐10 by administration of 2 mg JES5‐2A5 anti‐IL‐10 monoclonal antibody (mAb) 2h before LPS challenge resulted in a marked increase in both TNF and IFN‐γ serum levels while irrelevant isotype‐matched mAb had no effect. The enhanced production of inflammatory cytokines in anti‐IL‐10 mAb‐treated mice was associated with a 60% lethality after injection of 500 μg LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL‐10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.
Vaccination with T cell receptor peptides primes anti‐receptor cytotoxic T lymphocytes (CTL) and anergizes T cells specifically recognized by these CTL
Authors: Kuhröber Andreas , Schirmbeck Reinhold , Reimann Jörg
Published: March 2007

We selected three peptides from the germ‐line sequence of the Vβ8.2 and Jβ2.3 gene segments of the murine T cell receptor for antigen (TCR) which contained putative Kd‐ and Ld‐restricted epitopes. Immunization of BALB/c (H‐2d) mice with the Vβ8.2(67–90) 23‐mer peptide 1 as well as the 15‐mer Vβ8.2(95–108)‐peptide 2 efficiently primed specific CD8+ cytotoxic T lymphocyte (CTL) responses in vivo against natural TCR‐Vβ8.2 epitopes. Vβ8.2+ T cells were not deleted in TCR peptide‐immunized mice because the fractions of Vβ8.2+ CD4+ and Vβ8.2+ CD8+ T cells in spleen and lymph nodes were not altered. The proliferative response of Vβ8.2+ T cells to stimulation by monoclonal antibody F23.2 was selectively suppressed (by 60–80%) in peptide‐immunized BALB/c mice, indicating partial anergy of this T subset. Immunization of BALB/c mice with the Jβ2.3‐derived peptide 3 stimulated a CD8+ CTL response against a class I‐restricted epitope within this Jβ segment that was also generated during natural “endogenous” processing of this self antigen. These data confirm the predictive value of major histocompatibility complex class I allele‐specific motifs. The described experiments indicate that TCR peptide‐primed CD8+ CTL recognize class I‐restricted, natural Vβ/Jβ‐TCR epitopes. Such anti‐TCR CTL may, thus, operate in Vβ‐specific immunoregulation of the T cell system suppressing their functional reactivity without deleting them.
T cell allorecognition and endogenous HLA‐B27‐bound peptides in a cell line with defective HLA‐B27‐restricted antigen presentation
Authors: López Daniel , García‐Hoyo Rosa , García Fernando , De Castro José A. López
Published: March 2007

The B*2702+ lymphoblastoid cell line NW is unable to present at least some HLA‐B27‐restricted viral antigens to T cells. This defect was genetically inherited, and was suggested to be related to the nature of the HLA‐B27 binding peptides reaching the endoplasmic reticulum in these cells (Pazmany et al., J. Exp. Med. 1992. 175: 361). In the present study 17 of 19 HLA‐B27‐specific alloreactive cytotoxic T lymphocyte clones recognizing the B*2702 subtype on other cells also lysed NW cells. Only two cytotoxic T lymphocyte clones failed to lyse NW while efficiently killing other B*2702+ cell lines. The high‐performance liquid chromatography profiles of the B*2702+ bound peptides extracted from NW cells was similar, but not identical, to those from two other cell lines. These results indicate that the HLA‐B27‐bound peptide repertoire in NW cells is not fundamentally different from those in other B*2702+ cells. Our data argue against gross differences in peptide processing or transport as being responsible for the defective presentation of particular HLA‐B27‐restricted viral antigens to T cells, but do not rule out distinct presentation of some endogenous peptides. Differences in the capacity to present certain peptides could cause differential susceptibility among HLA‐B27+ individuals to ankylosing spondylitis.
Inhibition of anti‐GD3‐ganglioside antibody‐induced proliferation of human CD8+ T cells by CD16+ natural killer cells
Authors: Claus Christine , Schlaak Jörg , Dittmayer Maria , Zum Karl‐Hermann Meyer , Büschenfelde  , Dippold Wolfgang
Published: March 2007

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4+ and CD8+ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8+ and CD4+ T cells of normal donors containing less than 5% CD16+ natural killer (NK) cells. In contrast to CD4+ T cells, CD8+ T cells proliferated only weakly in the presence of 15% CD16+ NK cells. The proliferative response of purified CD4+ and CD8+ T cells (<5% NK cells) correlated with the antibody‐dependent induction of integral and soluble interleukin‐2 (IL‐2) receptors and was reduced to 20% by an anti‐IL‐2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4+ and CD8+ T cells and that CD16+ NK cells selectively inhibit anti‐GD3 antibody‐induced proliferation of CD8+ T cells.
CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells
Authors: Zupo Simona , Rugari Emanuela , Dono Mariella , Taborelli Giuseppe , Malavasi Fabio , Ferrarini Manlio
Published: March 2007

The present study demonstrates that an agonistic anti‐CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa‐stained cytospin preparations or flow cytometry on propidium iodidestained cells. Two other anti‐CD38 mAb (Leu‐17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up‐regulates the bcl‐2 proto‐oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti‐CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti‐CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti‐CD40, CD40L or recombinant interleukin‐4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.
Processing of viable group A streptococci leads to major histocompatibility complex class II presentation of T cell epitopes from the major protective antigen
Authors: Rossiter Beth A. , Alfonso Christopher , Kehoe Michael A. , Robinson John H.
Published: March 2007

We have previously mapped major histocompatibility complex (MHC) class II‐restricted T cell epitopes of the surface M protein of type 5 group A streptococci (M5) and show here that two out of four epitopes investigated were efficiently processed during incubation of viable streptococci with spleen cells for presentation to M5‐specific murine T cell clones. Viable streptococci were processed more efficiently than heat‐killed bacteria suggesting that secreted virulence factors of streptococci do not obstruct processing of streptococcal antigens in the dose range used. Epitopes from different regions of M5 could be ranked according to the efficiency with which they were processed, which may contribute to their relative immunodominance. It was further demonstrated that T cell clones specific for M5 308–319, an epitope from the M type conserved carboxy‐terminal half of M5, cross‐reacted between M5, M6 and M12, but not M49, streptococci. Helper T cell epitopes which are shared between streptococcal M types and are presented by MHC class II molecules on antigen‐presenting cells after processing of viable streptococci could be particularly useful in the design of multivalent streptococcal vaccines.
Comparing macrophages and dendritic leukocytes as antigen‐presenting cells for humoral responses in vivo by antigen targeting
Authors: Berg Siri Fuglem , Mjaaland Siri , Fossum Sigbjørn
Published: March 2007

Immunotargeting is a novel technique whereby antigen is directed against antigen‐presenting cells (APC) by conjugation to specific monoclonal antibodies (mAb). In this study we have employed the technique to investigate the efficiency of macrophages as APC compared with constitutively major histocompatibility complex (MHC) class II‐positive cells. i.e. dendritic leukocytes and B cells. in vivo. We first studied the organ retention of the radiolabeled conjugates by gamma counting, and their distribution within the draining lymph nodes by autoradiography. We could confirm that the conjugates reached the cells at which they were aimed. We then measured primary and secondary humoral responses. The results confirmed previous findings that targeting with mAb against MHC class II, i.e. to dendritic leukocytes, strongly enhanced the primary humoral response. In contrast, anti‐IgD conjugates, directed against B cells gave only weak primary responses. Although conjugates directed against macrophages were retained for a longer time than the other conjugates, the primary humoral response was virtually abolished. The secondary responses, however, were at least as strong as those obtained in animals primed with control conjugates, whereas animals primed with anti‐MHC class II conjugates showed little if any amplification of the secondary response. The discrepancies between the various conjugates could not be ascribed to TH1 versus TH2 responses as IgG1, IgG2a, IgG2b and IgE titers all co‐varied in single animals. A possible explanation for the observed results is that macrophages fail to induce cytokine production for terminal differentiation of B cells to plasma cells, whereas conversely, upon presentation by dendritic leukocytes most stimulated B cells mature to plasma cells, leaving less progeny for immunological memory.
Multiple natural killer cell‐activating signals are inhibited by major histocompatibility complex class I expression in target cells
Authors: Correa Isabel , Corral Laura , Raulet David H.
Published: March 2007

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggest that specific properties of the target cell other than the absence of class I expression are necessary to activate NK‐mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin‐2 activated NK cells, differ in their capacity to kill class I‐deficient lymphoblast target cells. In general, class I‐deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody‐dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK‐mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I‐mediated inhibition.
Characterization of brain‐isolated rat encephalitogenic T cell lines
Authors: Sun Deming , Hu Xian‐Zheng , Le Junyi , Swanborg Robert H.
Published: March 2007

In the present study, we have isolated and characterized five myelin basic protein (MBP)‐reactive T cell lines directly from the brains of Lewis rats during the early paralytic phase of experimental autoimmune encephalomyelitis (EAE). Each T cell line responded to the dominant encephalitogenic epitope spanning residues 68–88, and did not react against the conserved encephalitogenic epitope [MBP(87–99)] or the nonencephalitogenic MBP epitope [MBP(50–69)]. We determined the T cell receptor (TcR) β chain usage by polymerase chain reaction, DNA sequencing analysis and by generation of MBP‐reactive hybridomas from one of the T cell lines (BT74). The results revealed that brain‐infiltrating, MBP‐reactive T cells freshly isolated early in the course of the disease exhibit TcR diversity.
Signaling through CD50 (ICAM‐3) stimulates T lymphocyte binding to human umbilical vein endothelial cells and extracellular matrix proteins via an increase in β1 and β2 integrin function
Authors: Cid Maria C. , Esparza Jordi , Juan Manel , Miralles Agust́i , Ordi Jaume , Vilella Ramón , Urbano‐Márquez Alvaro , Gayà Antoni , Vives Jordi , Yagüe Jordi
Published: March 2007

Regulated adhesion of T lymphocytes to antigen‐presenting cells, endothelial cells and extracellular matrix proteins is crucial in T lymphocyte activation and migration to the sites of injury. In this study, we show that three monoclonal antibodies (mAb) recognizing different epitopes on the CD50 (ICAM‐3) molecule increase T lymphocyte adhesion to tumor necrosis factor (TNF)‐stimulated human umbilical vein endothelial cells and extracellular matrix proteins. These phenomena are mediated by an increase in β1 and β2 integrin avidity since (a) CD50‐induced adhesion to endothelial cells was abrogated by simultaneous blocking of β1‐ and β2‐mediated adhesion pathways but not by interfering with either one individually, (b) CD50 mAb increased β1 integrin‐mediated adhesion to extracellular matrix proteins and to fibronectin‐derived synthetic peptides, (c) CD50 mAb enhanced T lymphocyte binding to ICAM‐1 transfectants, and (d) CD50 mAb did not modify surface expression patterns of β1 or β2 integrins on T lymphocytes. Our data suggest that constitutively expressed CD50 (ICAM‐3) can play a pivotal role in initiating a cascade of adhesion events which may be crucial in immune activation and in the development of inflammatory lesions.
Suppression of polyclonal and antigen‐specific murine IgG1 but not IgE responses by neutralizing interleukin‐6 in vivo
Authors: Van Ommen René , Vredendaal Astrid E. C. M. , Savelkoul Huub F. J.
Published: March 2007

The crucial role of interleukin (IL)‐4 in the induction of murine IgG1 and IgE responses, which are coupled through the process of sequential isotype switching, has been well documented. Whereas IL‐4 is obligatory for the induction of IgE responses, it enhances IgG1 responses. In this study, using neutralizing antibodies, we provide evidence that, besides IL‐4, also IL‐6 is required for obtaining peak IgG1 responses. The mRNA levels of these two cytokines are coordinately expressed in the spleen of mice immunized with trinitrophenol‐keyhole limpet hemocyanin (TNP‐KLH). No IL‐6 requirement was observed for peak IgE responses. The IL‐6 dependence of IgG1 responses was found for both antigenspecific and polyclonal responses. Moreover, it was noted using TNP‐KLH and goat anti‐mouse (GAM) IgD as antigen that polyclonal IgG1 responses are more dependent on IL‐6 than antigen‐specific responses. In vitro experiments revealed that exogenous IL‐6 neither enhanced nor inhibited the IgG1 and IgE production by naive B cells, suggesting that IL‐6 did not interfere with the IL‐4‐induced isotype switch potential. Primary and memory IgG1 responses were both similarly dependent on IL‐6. These observations point to a role of IL‐6 in the terminal differentation of B cells switched to IgG1. Neutralization of IL‐6 did not inhibit either antigen‐specific or polyclonal IgE responses. Therefore, it was concluded that IL‐6 is not involved in the terminal differentiation of B cells switched to IgE. These findings thus provide a distinct role for IL‐6, besides IL‐4, in regulating murine IgG1 responses. The formation of IgE, however, is completely dependent on IL‐4 alone.
Induction of anergy in resting human T lymphocytes by immobilized anti‐CD3 antibodies
Authors: Wolf Hans , Müller Yvonne , Salmen Sibylle , Wilmanns Wolfgang , Jung Gundram
Published: March 2007

How the T cell receptor (TcR)/CD3 complex mediates not only the induction of T cell activation but also suppressive effects like T cell anergy or apoptosis is not well understood. Here we describe a series of preincubation and restimulation experiments which demonstrate that primary stimulation of resting, unseparated human T cells with mitogenic doses of immobilized anti‐CD3 antibodies induces hyporesponsiveness upon restimulation of the cells. Various costimuli can prevent this type of anergy to a variable degree if present during the preincubation period, phorbol 12‐myristate 13‐acetate (PMA) being the most and anti‐CD4 antibody the least effective. If employed together with anti‐CD3 antibody during the restimulation phase of the assay, interleukin (IL)‐2, IL‐4 and anti‐CD28 antibody break anergy almost completely. Proliferation induced by a submitogenic dose of anti‐CD3 antibody supplemented by costimulatory signals (anti‐CD2, anti‐CD4, anti‐CD28, IL‐2, IL‐4 or PMA) does not result in hyporesponsiveness. Taken together, these results support a modified view of the two‐signal model for T cell activation according to which anergy induction in resting T cells occurs if primary proliferation is induced by high density triggering of the TcR/CD3 complex in the absence of accessory signals. We discuss possible implications of these findings for the induction of peripheral tolerance.
Natural amino acid polymorphisms of the circumsporozoite protein of Plasmodium falciparum abrogate specific human CD4+ T cell responsiveness
Authors: Zevering Yinka , Khamboonruang Chirasak , Good Michael F.
Published: March 2007

Sequence polymorphism has been reported for virtually all malaria antigens and, in the case of the circumsporozoite (CS) protein, this variation is in the form of point mutations concentrated primarily in several regions recognized by T cells. The factors responsible for the variation are unknown. We studied the T cell responses to all known variants in malaria‐exposed Thais. Memory CD4+ T cells responded to variants of a polymorphic immunodominant region (denoted Th2R), and CD4+ T cell clones specific for one Thai Th2R variant were generated. There was minimal cross‐reactivity to any of the naturally occurring variants, including the other Thai variant, and competition studies performed with the clones using analog peptides demonstrated that all the substitutions of the polymorphic residues modulate either the binding of the peptide to major histocompatibility complex (MHC) molecules or the recognition by the T cell receptor of the peptide‐MHC complex. Our data suggest that CD4+ T cells may be able to select parasites expressing variant sequences and have implications for development of a CS‐based vaccine.
Interleukin‐10 prevents experimental allergic encephalomyelitis in rats
Authors: Rott Ortwin , Fleischer Bernhard , Cash Evelyne
Published: March 2007

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by myelin protein‐specific CD4+ T lymphocytes of the Th1‐like phenotype. In rats, the disease is characterized by a monophasic clinical manifestation, followed by a subsequent spontaneous remission and the establishment of life‐long resistance to reinduction of disease. Recent data indicate that intracerebral cytokine production, in particular synthesis of interleukin(IL)‐10, is selectively up‐regulated during the recovery phase of disease. This led us to assess the effects of IL‐10 on different rat lymphoid cell functions in vitro and to consider the possibility of an IL‐10‐mediated treatment to prevent the induction of central nervous system (CNS) autoimmune disease in vivo. Human recombinant IL‐10 suppressed interferon‐γ induced major histocompatibility complex class II up‐regulation in rat peritoneal macrophages, exhibited pleiotropic effects on thymocytes and totally abrogated tumor necrosis factor production of encephalitogenic T lymphocytes in vitro, without simultaneously affecting proliferative responses of the cells. Upon systemic administration during the initiation phase of disease, IL‐10 was effective in markedly suppressing the subsequent induction of EAE in Lewis rats. This suppression of clinical disease coincided with a significant and specific elevation of myelin basic protein‐specific autoantibody production, a sustained T cell proliferative response to myelin basic protein and a diminution of CNS infiltrations and thymic involutions in diseased animals. These data implicate IL‐10 as a possible candidate for treatment of Th1‐mediated CNS (auto‐) immune diseases.
Use of a skin test assay to determine tumor‐specific CD8+ T cell reactivity
Authors: Puccetti Paolo , Bianchi Roberta , Fioretti Maria C. , Ayroldi Emira , Uyttenhove Catherine , van Pel Aline , Boon Thierry , Grohmann Ursula
Published: March 2007

We have observed delayed‐type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I‐binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum−) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum− peptides resulted in a dose‐dependent DTH that peaked at approximately 24 h. The response was mediated by CD8+ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide‐pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2–6 months after priming. The evaluation by DTH in cancer patients of long‐lived CD8+ anti‐tumor T cell responses following local challenge with tumor‐specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.
A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice
Authors: Wallace Valerie A. , Kondo Seiji , Kono Takeshi , Xing Zhou , Timms Emma , Furlonger Caren , Keystone Edward , Gauldie Jack , Sauder Daniel N. , Mak Tak W. , Paige Christopher J.
Published: March 2007

The tight skin (Tsk/+) mouse represents a murine model of heritable fibrosis with some similarities to the skin fibrosis seen in human scleroderma. Tsk/+ animals display alterations in connective tissue in some internal organs. Skin fibrosis can be adoptively transferred to normal recipients with Tsk/+ bone marrow or spleen cells and older Tsk/+ animals develop autoantibodies against topoisomerase suggesting that some of the pathogenesis in the Tsk/+ mouse may be mediated by autoimmunity. To determine the role of T cell subsets in the pathogenesis of fibrotic disease, Tsk/+ mice were bred with CD4‐ and CD8‐deficient (CD4−/− and CD8−/−) mice. Tsk/+ CD4−/− mice showed a marked reduction in skin fibrosis as well as decreased cellularity and only mild collagen disorganization as compared to Tsk/+ CD4+ CD8+ control mice yet did not differ from Tsk controls in the level of serum anti‐topoisomerase activity. In contrast, Tsk/+ CD8−/− mice exhibited the same histology in the skin as Tsk/+ controls yet had significantly reduced levels of serum anti‐topoisomerase activity. Lung pathology, i.e. emphysema, was unaffected by both the CD4 or CD8 mutations. These data show that only some of the pathological effects of the Tsk mutation are T cell dependent and that different T cell subsets affect different parameters in this multi‐organ model of fibrotic disease.
Presentation of Listeria monocytogenes antigens by major histocompatibility complex class I molecules to CD8 cytotoxic T lymphocytes independent of listeriolysin secretion and virulence
Authors: Szalay Gudrun , Hess Jürgen , Kaufmann Stefan H. E.
Published: March 2007

Virulence and intracellular persistence of Listeria monocytogenes markedly depend on secretion of listeriolysin (Hly), which promotes invasion of the pathogen from the endosome into the cytosol. Recent studies have provided compelling evidence that Hly also facilitates recognition of listerial antigens, in association with major histocompatibility complex (MHC) class I molecules, by CD8 T lymphocytes. Data presented here confirm that the Hly‐deficient strains, the prfA− mutant L. monocytogenes SLCC53 and the transposon mutants L. monocytogenes M3 and M20 are avirulent for mice, and unable to replicate inside bone marrow‐derived macrophages (BMMΦ). Furthermore, BMMΦ infected with M3, M20 or SLCC53 were as efficiently lysed as BMMΦ infected with the Hly‐positive wild‐type strain EGD by MHC class I‐dependent CD8 cytotoxic T lymphocytes. Using the highly sensitive polymerase chain reaction method, hly mRNA was detectable in BMMΦ infected with L. monocytogenes EGD or SLCC53, but totally absent in M3‐infected BMMΦ. In the case of M20, an excision of the transposon occurred, but the excision was not precise and the hly gene was approximately 400 base pairs shorter. These findings argue against a unique role for Hly in MHC class I presentation of listerial antigens, although Hly appears central to virulence and intracellular replication. Thus, virulence of L. monocytogenes is dissociable from MHC class I presentation of listerial antigens.
CD57+ T lymphocytes are derived from CD57− precursors by differentiation occurring in late immune responses
Authors: d'Angeac Arnaud Dupuy , Monier Serge , Pilling Darrell , Travaglio‐Encinoza Adolfo , Rème Thierry , Salmon Mike
Published: March 2007

CD3+ T cells expressing the 110‐kDa CD57 antigen are found in survivors of renal, cardiac and bone marrow transplants, in patients with acquired immune deficiency syndrome and in patients with rheumatoid arthritis. They are also present in normal individuals and expand upon ageing. They do not grow in culture and their role in the immune response is poorly understood. The expression of the various isoforms of the leukocyte common antigen (CD45) identifies a spectrum of differentiation in CD4+ and CD8+ T cells ranging from naive (CD45RA+CD45RBbrightCD45RO−) through early primed cells (CD45RA−RBbrightROdull) to highly differentiated memory cells which are CD45RA−RBdullRObright. CD45 isoforms expressed by CD57+ T cells showed distinct differences between CD4+ and CD8+ populations, but in each case indicated an advanced state of differentiation. The expression of T cell receptor Vβ families was highly variable between individuals, but both CD57+ and CD57− cells show a full range of the specificities tested. Vβ expression was more closely related within either the CD4+ or the CD8+ subsets, irrespective of CD57 expression, than between these subsets, suggesting a relationship between CD57+ and CD57− cells within the same T cell pool. This possibility was supported by experiments showing that CD3+CD57+ lymphocytes were similar to CD3+CD57− T cells in terms of the production of basic T cell cytokines [interleukin (IL)‐2, IL‐4, and interferon‐γ]. Furthermore, in vitro stimulation of CD3+CD57− T cells in secondary mixed leukocyte reaction or by co‐culture with IL‐2 and IL‐4 induced the appearance of CD3+CD57+ cells with phenotypic and functional similarities to in vivo CD3+CD57+ cells. These data strongly suggest that the expression of CD57 is a differentiation event which occurs on CD57− T cells late in the immune response.
Immunoglobulin isotype regulation by antigen‐presenting cells in vivo
Authors: de Becker Geneviève , Sornasse Thierry , Nabavi Nasrin , Bazin Hervé , Tielemans Françoise , Urbain Jacques , Leo Oberdan , Moser Muriel
Published: March 2007

The isotype and magnitude of the B cell response clearly depends on the in vivo activation of T helper (Th) cells which secrete different lymphokines. Since Th are activated by the presentation of the antigen on specialized cells, we wished to test whether the nature of the antigen‐presenting cells (APC) influences the isotypic profile of the humoral response. Data are presented showing that antigen‐pulsed dendritic cells (DC) and peritoneal macrophages induce the synthesis of specific antibodies when injected in syngeneic animals. By contrast, a single injection of antigen‐pulsed resting B cells does not prime the mice in vivo. Moreover, the injection of antigen‐pulsed DC induces the synthesis of specific IgG2a and IgG1 antibodies, whereas peritoneal macrophages favor the production of IgG1 and IgE antibodies specific for the antigen. These data show that the isotype and the amplitude of the B cell response can be regulated by the nature of the APC, and indirectly suggest that Th cell differentiation is controlled at the level of antigen presentation.
Deletion of T lymphocytes in human CD4 transgenic mice induced by HIV‐gp120 and gp120‐specific antibodies from AIDS patients
Authors: Wang Zhi‐Qin , Orlikowsky Thorsten , Dudhane Anita , Mittler Robert , Blum Michelle , Lacy Elizabeth , Riethmüller Gert , Hoffmann Michael K.
Published: March 2007

CD4, a T cell receptor for major histocompatibility complex class II antigen, is a key regulator of immunological reactivities. When engaged together with the T cell antigen receptor, CD4 enhances immune reactions, whereas when ligated independently of the antigen receptor CD4 inhibits the activation of T cells or initiates their deletion. CD4 serves also as a receptor for the human immunodeficiency virus (HIV), which binds the receptor with high avidity through its envelope molecule, gp120. Studies in tissue culture have shown that its affinity to CD4 gives the virus opportunities to utilize CD4‐mediated signaling and to manipulate immunocytes. We show here in human CD4 transgenic mice that appropriately cross‐linked HIV envelope protein causes massive deletion of HIV‐reactive T cells in vivo.