Biomedical subjects

J M Kanellopoulos

Publications and source records attributed to J M Kanellopoulos.

14 recordsLinked to original sources

PubMed1999

P2Z/P2X7 receptor-dependent apoptosis of dendritic cells.

Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.

Adenosine Diphosphate↗

PubMed1998

Internalization of Chlamydia by dendritic cells and stimulation of Chlamydia-specific T cells.

Chlamydia species are the causative agents of trachoma, various forms of pneumonia, and the most common sexually transmitted diseases. Although the infection cycle has been extensively characterized in epithelial cells, where the Chlamydia entry-vacuoles avoid fusion with host-cell lysosomes, the cellular immune response has received less attention. Moreover, despite the abundant presence of dendritic cells (DC) in the sites of infection, the interaction between Chlamydia and DC has never been studied. We observe that DC kill Chlamydia trachomatis and Chlamydia psittaci. The chlamydiae are internalized by the DC in a nonspecific manner through macropinocytosis, and the macropinosomes fuse subsequently with DC lysosomes expressing MHC class II molecules. The interaction induces maturation of the DC, since presentation of an exogenous Ag is severely inhibited after a 1-day incubation, although chlamydial Ags are still presented and recognized by Chlamydia-specific CD4+ T cells. Thus, DC most likely play a role in initiating the T cell response in vivo and could potentially be used in adoptive transfer therapies to vaccinate against Chlamydia.

Animals↗

PubMed1997

Determinant selection for T-cell tolerance in HEL-transgenic mice: dissociation between immunogenicity and tolerogenicity.

The induction of T-cell tolerance to self-antigens has been extensively characterized for immunodominant (ID) regions. However, tolerance toward other minor self-determinants has received less attention. In the H-2(d) haplotype, HEL contains a single ID determinant (region 102-120) presented by I-E(d) MHC class II molecules. The present study evaluates the role of subdominant and cryptic HEL regions in maintaining tolerance. We have generated a mutated HEL antigen, HEL mu, whose ID region does not bind to I-E(d). Lymph node cells from HEL-immunized mice proliferated strongly to HEL mu in vitro. Two new stimulatory regions common to HEL and HEL mu were uncovered. They are produced during antigen processing and prime specific T lymphocytes. HEL-Tg mice were tolerant to these determinants, thus confirming their in vivo presentation. These HEL regions were as tolerogenic as the HEL ID determinant, despite their poor immunogenicity. These results demonstrate that there is not always a correlation between tolerogenicity and immunogenicity, a finding that may be critical for understanding T-cell tolerance.

Animals↗

PubMed1994

Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice.

We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.

Amino Acid Sequence↗

PubMed1994

Dose-dependent T cell tolerance to an immunodominant self peptide.

We have previously described a model of tolerance to self peptides in a mouse transgenic (Tg) line producing secreted hen egg-white lysozyme (HEL). The HEL cDNA was placed under the control of a ubiquitous promoter expressed early in embryogenesis, so that HEL should be present in Tg mice throughout the development of the immune system. Since individual HEL Tg mice express different amounts of serum HEL, we were previously able to show that H-2d mice with HEL blood level > 10 ng/ml are tolerant to HEL and to the immunodominant (ID) peptide 108-116. However, autoreactive T lymphocytes recognizing the HEL subdominant (SD) peptides 74-96 and 1-18 still persist and the SD-specific response disappear at higher blood HEL concentrations. In the present work, we have studied HEL Tg H-2d mice with HEL serum levels < 10 ng/ml (HEL-low Tg animals). We find that 50% of Tg animals with HEL blood concentration < 2 ng/ml are responsive to HEL in T cell proliferation assays, although these responses are lower than those seen in non-Tg control mice. The HEL-specific T lymphocytes react only with 15-mer overlapping peptides encompassing the single H-2d ID region of HEL (residues 102-122); whereas the 9-mer minimal ID peptide 108-116, which strongly triggers non-Tg T cells, is unable to stimulate auto-reactive T cells in vitro from HEL-low Tg mice. Altogether, our results suggest that T lymphocytes specific for the minimal ID peptide are deleted or inactivated, while T cell clones of lower affinity and reacting with epitopes on longer peptides persist. Thus, the high affinity ID peptide-specific T cell clones can be negatively selected even in the presence of low amounts of HEL.

Amino Acid Sequence↗

PubMed1994

Is antigen processing guided by major histocompatibility complex molecules?

Major histocompatibility complex class I (MHC-1) molecules bind peptide fragments derived from cytosolic antigens, and class II (MHC-2) molecules bind fragments of proteins that enter the endocytic pathway. How peptides of the right affinity and size are generated in vivo is still the focus of intense research. Current data are consistent with the view that precursor peptides of varying length are produced in the cytosol and transported into the endoplasmic reticulum lumen where nascent MHC-1 could sample the peptides for their affinity. High-affinity peptides would form stable complexes with MHC-1, which are resistant to proteolysis by luminal enzymes; peptides unable to bind to MHC-1 presumably undergo proteolysis in the lumen. On the other hand, multiple mechanisms are probably used to load MHC-2. Some proteins denatured in the acidic and reducing environment of the endosomes most likely bind to MHC-2 through the antigen's immunodominant region, and the exposed portions of the antigen are degraded by endosomal proteases. Other antigens must first be proteolysed into peptide fragments, which compete among themselves for binding to MHC-2, whereas heat shock proteins could also contribute peptides for MHC-2 loading. Because of their respective loading modes, there is a partial correlation between the MHC-2 binding affinity of the protein fragments and their in vivo immunodominance, which may not necessarily be the case for MHC-1.

Antigen Presentation↗

PubMed1992

Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants.

We have produced transgenic mice expression hen egg-white lysozyme (HEL) under the control of a ubiquitous promoter, so that in transgenic animals, HEL is presumably present in the serum and thymus throughout the period of establishment of the T-cell repertoire. We show that HEL transgenic H-2d mice with HEL blood levels greater than 10 ng/ml are tolerant to HEL as well as to the immunodominant peptide 108-116. Thus, their T lymphocytes do not proliferate in response to the immunodominant peptide 108-116 after in vivo immunization with HEL or peptide 108-116. In contrast, in transgenic mice tolerant to HEL, the state of tolerance to subdominant peptides 1-18 and 74-96 appears variable and highly depended on HEL blood levels. Complete unresponsiveness is seen when HEL serum levels are high, and this unresponsiveness is reached at a lower HEL concentration for peptide 1-18 than for peptide 74-96. Thus, a hierarchy exists among the three peptides (108-116 much greater than 1-18 greater than 74-96) for induction of a response to HEL and for HEL tolerance induction in T cells specific for these peptides. Persistence in the periphery of autoreactive T cells recognizing subdominant peptides of self-proteins, as shown in this transgenic model, indicates that self-tolerance is limited to a subset of dominant self-peptides and suggests a role for T lymphocytes specific for subdominant determinants in autoimmunity.

Animals↗

PubMed1989

T-cell chemiluminescence. A novel aspect of T-cell membrane activation studied with a Jurkat tumour cell line.

The binding of mitogenic lectins phytohaemagglutinin (PHA), concanavalin A (Con A) and/or of monoclonal antibodies to different receptors such as antigen receptor complex or CD2 on human T cells generates increases in the concentrations of inositol triphosphate (IP3) and cytoplasmic free calcium. This T lymphocyte requires the delivery of two signals; the first can be provided by specific monoclonal antibodies or by mitogenic lectins, and the second by a phorbol ester, phorbol myristate acetate (PMA). In other cells such as macrophages, the rise of intracellular calcium via the generation of IP3 and stimulation of protein kinase C can activate the phospholipase A2, a calcium-dependent enzyme. This enzyme initiates the release of reactive oxygen intermediates and metabolites of arachidonic acid. In order to know whether this other metabolic pathway can be generated in T cells, we tested the capacity of different T-cell lines and clones to produce superoxide anion after stimulation by the above-mentioned activating agents. In this paper, we demonstrate that treatment of the Jurkat human cell line with Con A, PHA, and PMA results in a significant release of reactive oxygen metabolites. Of the various T-cell lines and clones tested, only Jurkat exhibited an oxidative burst. Moreover, none of the antibodies tested (anti-CD3, anti-CD2, and anti-CD28) and known to activate T cells, and none of the immune complexes was able to mediate such an effect. The existence of an oxidative metabolism in at least one T-cell line suggests that T-cell activation may in some instances use another metabolic pathway.

Free Radicals↗

PubMed1987

Interferon-gamma restores T lymphocyte proliferation of nonresponders to IgG1 anti-CD3 via the induction of Fc gamma 1 receptors on monocytes.

Human peripheral blood T lymphocytes are stimulated to grow and divide by some mouse anti-CD3 monoclonal antibodies. This polyclonal mitogenesis is dependent on both their immunoglobulin subclass and the presence of monocytes. The unresponsiveness of T lymphocytes from certain individuals to mouse IgG1 (or IgG2a) antibodies is due to a failure of their monocytes to bind these IgG isotypes. In this study, we have selected such nonresponder subjects to IgG1 anti-CD3 (UCHT 1) in order to study their monocytes. Two assays were used: IgG1 and IgG2 EA rosettes to evaluate their Fc receptor-binding capacity, and IgG-mediated monocyte chemiluminescence to test their receptor-related activation since mouse anti-T cell antibodies binding to lymphocytes trigger monocyte chemiluminescence via their Fc receptor. We have observed that in all nonresponder subjects the absence of IgG1 anti-CD3 monocyte chemiluminescence strictly correlates with the absence of IgG1 EA rosettes. Thus, the failure to respond to UCHT 1, in all nonresponders tested to date, is due to the absence of Fc gamma 1 receptors on their monocytes. Treatment of nonresponder monocytes by recombinant interferon-gamma was shown to restore T cell proliferation and monocyte chemiluminescence in nonresponders. This effect of interferon-gamma correlates with the appearance of Fc gamma 1 receptors on monocytes from these individuals. This work strongly suggests that nonresponder monocytes possess functional genes for Fc gamma 1 receptors which are not expressed normally at a detectable level but can be induced by interferon-gamma.

Antibodies, Monoclonal↗

PubMed1986

The human cell surface glycoprotein complex (gp 120,200) recognized by monoclonal antibody K20 is a component binding to phytohaemagglutinin on T cells.

Monoclonal antibody K20 recognizes a human glycoprotein complex that is not restricted to haematopoietic lineages but is preferentially expressed on early haematopoietic cells, T cells, and monocytes. This glycoprotein complex is made of a constant 120,000-140,000 Mr subunit noncovalently associated at the cell surface with subunits of higher Mr ranging from 150,000 to 200,000 on different cell types. Internal labelling with [35S]methionine and pulse-chase experiments revealed that in the cell the 120,000 Mr glycoprotein of this complex is also noncovalently associated with a 100,000 Mr glycoprotein, and that both glycoproteins are independently biosynthesized. This glycoprotein complex is shown by immunoprecipitation by lectin plus antilectin antibodies and by sequential immunoprecipitations to be one of the cell surface structures bound by phytohaemagglutinin on the surface of normal T cells.

Animals↗

PubMed1986

The sheep erythrocyte receptor and both alpha and beta chains of the human T-lymphocyte antigen receptor bind the mitogenic lectin (phytohaemagglutinin) from Phaseolus vulgaris.

We have studied the interaction of mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A) with both surface molecules which, by the use of monoclonal antibodies, are known to trigger T-cell mitogenesis. Monoclonal antibodies recognizing the T-lymphocyte receptor for antigen (Ti) and/or its associated structure, CD3, activate T cells. More recently, a second pathway of activation has been described which involves the sheep erythrocyte binding glycoprotein CD2, a surface molecule distinct from Ti-CD3. Lysates from surface-iodinated T-leukaemia cell lines were treated with lectin and affinity purified anti-lectin antibodies coupled to protein A-Sepharose. We have shown that eluates from Con A/anti-Con A or PHA/anti-PHA immunoprecipitates contained Ti, since a rabbit anti-T alpha serum, which recognizes the native and denatured forms of the constant region of the alpha chain, immunoprecipitated Ti from these eluates. Furthermore, Ti immunoprecipitated by anti-T alpha serum from lysates of surface iodinated E+ lymphocytes was binding to PHA after elution from the immunoprecipitate. When the purified Ti molecule was reduced and alkylated, allowing the permanent dissociation of its alpha and beta subunits, PHA interacted with both chains, whereas anti-T alpha serum immunoprecipitated the alpha chain only. Altogether, these results demonstrate that PHA interacts with both chains of the T cell receptor for antigen on human peripheral T lymphocytes. With the HPB-ALL tumour line, a similar approach showed that both alpha and beta chains of Ti bind to Con A and Ulex europaeus 1 but not Helix pomatia. Affinity chromatography on immobilized lectins and immunoprecipitation with lectin/anti-lectin antibodies were employed to test whether CD2 binds to PHA and Con A. The results show that CD2 from human peripheral T lymphocytes binds both lectins but with a lower affinity for PHA than Con A.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1985

The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes.

Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).

Antibodies, Monoclonal↗

PubMed1983

Biosynthesis and molecular nature of the T3 antigen of human T lymphocytes.

Immunoprecipitates of the T3 antigen prepared from HPB-ALL cells by using the monoclonal antibody UCH-T1 were analysed by SDS-polyacrylamide gel electrophoresis. Cells which had been biosynthetically labelled for up to 4 h gave a major polypeptide of mol. wt. 19 000 plus two weaker, more diffuse bands of mol. wts. 21 000 and 23 000, whereas surface labelled cells gave a prominent band of mol. wt. 19 000, a major band of 21 000 and a weaker diffuse band of approximately 26 000. As judged from their sensitivity to proteinase-K digestion, all the above polypeptides possess a transmembrane orientation. Digestion with endoglycosidases H and F (endo-H and endo-F), and tunicamycin treatment indicate that all the polypeptides, except that of 19 000 mol. wt. are N-glycosylated. The 21 000 and 23 000 mol. wt. chains possess both immature and mature oligosaccharide units, whereas the 26 000 mol. wt. band apparently has mature units only. Pulse chase experiments combined with digestion by endo-F and endo-H suggest that the N-glycosylated polypeptides are derived from two polypeptides of mol. wts. 14 000 and 16 000. It is concluded that the T3 antigen is derived from three different non-glycosylated polypeptides two of which are subsequently N-glycosylated to give the 21 000, 23 000 and 26 000 forms. The cell surface T3 antigen most probably comprises at least two distinct, non-covalently associated polypeptides, but the number and types of polypeptides giving rise to the whole molecule and whether different complexes exist is at present unclear.

Antibodies, Monoclonal↗

PubMed1980

Composition and subunit structure of the cell receptor for immunoglobulin E.

The principal surface glycoprotein which specifically binds immunoglobulin E was isolated from rat basophilic leukemia cells in sufficient amounts for compositional and end group analyses. The protein has about 30% carbohydrate and a relatively low content of hydrophobic amino acid residues. No NH2-terminal residue was found by standard methods. The data suggest a Mr approximately equal to 50,000. The latter value is calculated on the basis of 1 molecule of receptor binding 1 molecule of immunoglobulin E. New data confirmed this valence. We propose a provisional model in which the principal component which binds immunoglobulin E is a monomer which, in cells and in nondenaturing solvents, is associated in a 1:1 ratio with the polypeptide of Mr approximately equal to 30,000 recently defined by studies employing cross-linking reagents.

Amino Acids↗