Biomedical subjects

P Portolés

Publications and source records attributed to P Portolés.

12 recordsLinked to original sources

PubMed2000

Crry/p65, a membrane complement regulatory protein, has costimulatory properties on mouse T cells.

It is known that certain type I membrane molecules (complement receptors type 1 and 2) belonging to the regulators of complement activation (RCA) family are involved in the regulation of B lymphocyte activation. In contrast, only GPI-anchored RCA molecules (CD55) have been described to be involved in T lymphocyte activation. In this study, we describe a novel function for the mouse RCA type I membrane protein Crry/p65 as a costimulatory molecule in CD4+ T cell activation. This is shown by increased anti-CD3-induced proliferation of CD4+ spleen T lymphocytes in the presence of the Crry/p65-specific mAb P3D2. Furthermore, Ab-induced coligation of Crry/p65 and CD3 favors IL-4 rather than IFN-gamma secretion in these cells. Crry/p65 signaling was also observed regardless of additional Ca2+, protein kinase C, or CD28-mediated costimuli. Analysis of intracellular intermediaries shows that Crry/p65-CD3 coligation enhances certain TCR/CD3-mediated signals, producing increased early tyrosine phosphorylation of many substrates and enhanced activation of the mitogen-activated protein kinase, extracellular signal-related kinase. These data fit well with the association of Crry/p65 with the tyrosine kinase Lck found in T cell lysates. The epitope recognized by the mAb P3D2 interferes with the protective role of Crry/p65 on C3 deposition. The relationship between protective function and costimulation by Crry/p65 is discussed. Our results support a multifunctional role for Crry/p65 in T cells and suggest new links between the natural and adaptive immune responses.

Adjuvants, Immunologic↗

PubMed2000

Variability of invariant mouse CD3epsilon chains detected by anti-CD3 antibodies.

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.

Alleles↗

PubMed1999

Antibody-induced CD3-CD4 coligation inhibits TCR/CD3 activation in the absence of costimulatory signals in normal mouse CD4(+) T lymphocytes.

The effect of CD3-CD4 coligation on CD3-mediated activation of normal mouse CD4(+) T lymphocytes has been analyzed in the absence of exogenous lymphokines. If anti-CD3 and anti-CD4 antibodies are adsorbed to culture wells by means of previously adsorbed anti-Ig antibodies (indirect binding), CD3-CD4 coligation inhibits activation measured as cell proliferation or as secretion of IL-2, IL-4, and IFN-gamma. Addition of IL-2, anti-CD28 antibodies, or phorbol esters, but not IL-1, IL-4, or ionomycin, blocked CD4-mediated inhibition and restored the response to levels equal or higher than those of cultures activated by anti-CD3 alone. In contrast, CD3-CD4 coligation by antibodies directly adsorbed to culture wells potentiated anti-CD3-induced activation, either in the absence or in the presence of exogenous costimuli. Similar results were observed when CD4(+) T cells of naive phenotype (CD44(low), CD45RB(high)) were used in the experiments. The analysis of early tyrosine phosphorylation in CD4(+) T cells shows that phosphorylation of many cell substrates is clearly enhanced upon CD3-CD4 coligation using indirectly or directly bound antibodies, yet certain substrates are mainly phosphorylated under inhibitory conditions. Although CD28 ligation does not produce any clear change in the tyrosine phosphorylation pattern in lysates from cells activated by indirectly bound anti-CD3 plus anti-CD4 antibodies, the analysis of active forms of the MAP kinase ERK suggests that downstream signaling pathways involved in IL-2 gene activation can be differentially activated depending on the direct or indirect CD3-CD4 adsorption and CD28 ligation.

Adsorption↗

PubMed1998

NF-kappaB in Th2 cells: delayed and long-lasting induction through the TCR complex.

Nucelar NFkappaB was analyzed in murine Th2 cells after stimulation via the TCR pathway. Signals delivered through the TCR/CD3 complex induced active NFkappaB translocation to the nucleus of Th2 cells after a late phase (24 h) of the activation process, which is in contrast to the rapid appearance of nuclear NFkappaB (3 h) in Th1 cells after the same stimulation. The slow kinetic of NFkappaB nuclear uptake in Th2 cells was not accelerated by CD28 triggering or under stimulation with antigen plus antigen-presenting cells. Th1 and Th2 cells were also different in the composition of NFkappaB complexes induced. Whereas in Th1 cells TCR triggering induced the presence of nuclear p50.p65 heterodimers, in Th2 cells the complexes induced were shown to be composed of p65 plus another NFkappaB protein distinct from p50. The delayed NFkappaB induction in Th2 cells was dependent on protein synthesis and the significance of this is discussed.

Animals↗

PubMed1997

CD4 dependence of activation threshold and TCR signalling in mouse T lymphocytes.

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.

Animals↗

PubMed1996

Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor.

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single-positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide-linked dimer, precipitating as a broad band at about 50-65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N-glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross-blocking, co-capping, co-modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co-stimulatory molecules such as CD69, CD2, Ly-6, CD25, OX-40, Mac-1 and LFA-1. By in vitro kinase assay, H4 was found to co-precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co-modulation and co-capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell-specific co-stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.

Animals↗

PubMed1995

A hyperreactive variant of a CD4+ T cell line is activated by syngeneic antigen presenting cells in the absence of antigen.

A variant of the murine CD4+ T helper cell clone D10.G4.1 (D10) has been isolated and cloned. This line, which we have named "syngeneic-reactive D10", or SR.D10, maintains the I-Ak-restricted specificity for Conalbumin and the allogeneic specificities characteristic of D10 cells. However, it is hyperreactive to TCR-dependent and-independent stimuli, indicating a lower activation threshold than the original D10.G4.1 clone. The hyperreactivity of SR.D10 runs in parallel with the acquisition of a reactive phenotype against syngeneic antigen presenting cells (APCs). As in antigen activation, reactivity to syngeneic APCs can be inhibited by anti-TCR, anti-CD4, or anti-class II monoclonal antibodies. The role of CD4 in this phenomenon is highlighted as "syngeneic reactivity" disappears in CD4- mutants of SR.D10 and is recovered in CD4 transfectants. The expression of several cell surface molecules involved in T cell activation show qualitative and/or quantitative differences between SR.D10 and the original D10. No significant differences in quantity and activity of p56lck and p59fyn were detected between the hyperreactive and the original clone. Our results suggest that high sensitivity to activation, concomitant with expression of CD4, might allow the acquisition of an autoreactive phenotype and confirm the important contribution of coreceptors to determine the activation threshold of the cells. The characteristics of SR.D10 and the possibility of growing them in the presence of interleukins make this cell line a experimental model of great interest for analyzing activation mechanisms in T cells.

Animals↗

PubMed1994

Genetic and immunochemical evidence for CD4-dependent association of p56lck with the alpha beta T-cell receptor (TCR): regulation of TCR-induced activation.

Recent observations suggest that the tyrosine kinase p56lck is involved in the transduction of transmembrane signals through the antigen specific T cell receptor (TCR) in CD4+ T cells. By means of in vitro kinase assays, we have found that p56lck coprecipitated with the TCR from lysates of a murine CD4+ T cell line in the absence of TCR-mediated stimuli. Analysis of CD4- mutants and CD4-transfected cells shows that p56lck-TCR association occurred only when CD4 was present. The functional importance of CD4:p56lck-TCR association was demonstrated by low activating potential of rare clonotypic antibodies which did not coprecipitate CD4:p56lck, as well as by total or partial loss of anti-TCR or antigen induced stimulation in CD4- cells, which could be recovered by CD4 transfection. Complementation assays using different anti-TCR antibodies suggest that cross linking of TCR-p56lck:CD4 plus structural changes in the complex are needed for efficient transduction of activating signals through the TCR in these cells.

Animals↗

PubMed1994

Polyerga, a biological response modifier enhancing T-lymphocyte-dependent responses.

Cancer patients are often treated with biological response modifiers to enhance immunological functions. However, little is known about the actual mechanism of action of many of these substances. Therefore, we were interested in the effect of i.p. treatment with porcine low-molecular-weight spleen peptides, which are used during supportive cancer therapy, on lymphoid cell populations and function in mice. After treatment with 0.5 microgram peptides/kg body weight for 14 consecutive days, lymphokine secretion and the generation of cytotoxic T-cells were significantly enhanced as compared with controls. However, there was no effect on the number of cells or the percentage of cells expressing functional surface markers in secondary lymphoid organs.

Animals↗

PubMed1990

A simple method for the radioactive iodination of CD4 molecules.

A method for the vectorial radioiodination of CD4 and other membrane proteins having few or no tyrosine residues in the extracytoplasmic domains is described. Incubation of the cells with sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP), a water-soluble derivative of the Bolton-Hunter reagent results in the coupling of hydroxyphenyl groups to free amino groups of cell surface proteins and these groups are then vectorially radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The method is highly efficient, gentle, fast, simple, and does not require the previous radiolabelling of the Bolton-Hunter reagent.

Animals↗

PubMed1986

Enhancement of lymphocyte proliferation, interleukin-2 production and NK activity by inmunoferon (AM-3), a fungal immunomodulator: variations in normal and immunosuppressed mice.

The in vivo effect of Inmunoferon (AM-3), a glycophosphopeptide of fungal origin, has been studied on T and B lymphocyte mitogenesis, Interleukin 2 (IL-2) synthesis and natural killer (NK) activity. Inmunoferon (30 mg/kg/day) was administered to several groups of mice 2, 3 or 7 days/week for 2 weeks, and its effect assessed on day 15 of treatment. Every treatment assayed enhanced IL-2 and NK activity in the spleen. The greater effect was produced by daily administration of the immunomodulator. No enhancement was found in mitogen-induced proliferation of T and B lymphocytes. Similar treatments with Inmunoferon enhanced NK activity in the spleens of mice treated with 250 mg/kg cyclophosphamide. In addition, mitogenic responses of T lymphocytes, but not IL-2 production, were also increased in immunosuppressed mice after treatment with the immunomodulator.

Animals↗

PubMed1984

Inhibitory mechanisms of 5-fluorodeoxyuridine on mitogen-induced blastogenesis of lymphocytes.

5-Fluorodeoxyuridine inhibited DNA synthesis of mitogen-stimulated T and B cells as determined by either incorporation of 3H-thymidine to the cells or measure of DNA content in the cells by microdensitometry. 5-Fluorodeoxyuridine concentrations necessary to inhibit DNA synthesis by 50% in the stimulated cultures were high (2 micrograms/ml and 7 micrograms/ml for B and T cells, respectively). Addition of exogenous thymidine (100 micrograms/ml) partially reversed the inhibition of DNA synthesis by 5-fluorodeoxyuridine (10 micrograms/ml). These data indicate that lymphocytes possess a mechanism of resistance to inhibition of thymidilate synthetase by 5-fluorodeoxyuridine. On the other hand, the inhibition of mitogen-induced blastogenesis is not only mediated by an effect on thymidilate synthetase but could also be mediated by other mechanisms, such as inhibition of nucleic acid precursor transport into the cells.

Animals↗