375 research outputs found

    T-cell antigenic sites tend to be amphipathic structures.

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    Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL

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    Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether, such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-α release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell

    Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

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    Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation. A common theme that is emerging from the study of tumor-associated translocations is the generation of chimeric genes that, when expressed, frequently retain many of the functional properties of the wild-type genes from which they originated. Sarcomas, in particular, harbor chimeric genes that are often derived from transcription factors, suggesting that the resulting chimeric transcription factors contribute to tumorigenesis. The tumor-specific expression of the fusion proteins make them likely candidates for tumor-associated antigens (TAA) and are thus of interest in the development of new therapies. The focus of this review will be on the translocation events associated with Ewing's sarcomas/PNETs (ES), alveolar rhabdomyosarcoma (ARMS), malignant melanoma of soft parts (MMSP) (clear cell sarcoma), desmoplastic small round cell tumor (DSRCT), synovial sarcoma (SS), and liposarcoma (LS), and the potential for targeting the resulting chimeric proteins in novel immunotherapies

    Structural comparison of a 15 residue peptide from the V3 loop of HIV-1IIIb and an O-glycosylated analogue

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    AbstractAs part of a program to study the effect of glycosylation on the three-dimensional structures of HIV-1IIIB V3 peptide constructs, we have examined the solution structures of a 15 residue peptide (RIQRGPGRAFVTIGK, P18IIIB), originally mapped as an epitope recognized by CD8+ Dd class I MHC-restricted murine cytotoxic T-lymphocytes (CTL), and an analogue (P18IIIB-g), O-glycosylated with an α-galactosamine on Thr-12, using NMR, circular dichroism and molecular modeling methods. Our studies show that the peptides sample mainly random conformations in aqueous solution near 25°C and become more ordered by the addition of trifluoroethanol. Upon decreasing the temperature to 5°C, a reverse turn is formed around the immunodominant tip (G5−R8). Glycosylation on T12 ‘tightens’ the turn slightly as suggested by NOE and CD analysis. In addition, the sugar has a defined conformation with respect to the peptide backbone and influences the local peptide conformation. These data suggest that simple glycosylation may influence the conformational equilibrium of a V3 peptide which contains a domain critical for antibody recognition and virus neutralization. We also show that the ability of cytotoxic T-lymphocytes (CTL) to lyse tumor cells presenting P18IIIB was completely abrogated by threonine glycosylation

    Inactivation of human immunodeficiency virus (HIV)-1 envelope-specific CD8+ cytotoxic T lymphocytes by free antigenic peptide: A self-veto mechanism?

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    Free peptide has been found to inhibit cytotoxic T lymphocyte (CTL) activity, and veto cells bearing peptide-major histocompatibility complex (MHC) complexes have been found to inactivate CTL, but the two phenomenon have not been connected. Here we show that a common mechanism may apply to both CD8+ CTL lines or clones specific for a determinant of the human immunodeficiency virus (HIV)-1 IIIB envelope protein gp160, P18IIIB, are inhibited by as little as 10 min exposure to the minimal 10-mer peptide, 1- 10, within P18IIIB, free in solution, in contrast to peptide already bound to antigen-presenting cells (APC), which does not inhibit. Several lines of evidence suggest that the peptide must be processed and presented by H-2D(d) on the CTL itself to the specific T cell receptor (TCR) to be inhibitory. The inhibition was not killing, in that CTL, did not kill 51Cr-labeled sister CTL in the presence of free peptide, and in missing experiments with CTL lines of different specificities restricted by the same MHC molecule, D(d), the presence of free peptide recognized by one CTL line did not inhibit the activity of the other CTL, line that could present the peptide. Also, partial recovery of activity could be elicited by restimulation with cell-bound peptide, supporting the conclusion that neither fratricide nor suicide (apoptosis) was involved. The classic veto phenomenon was ruled out by failure of peptide-bearing CTL to inactivate others. Using pairs of CTL lines of differing specificity but similar MHC restriction, each pulsed with the peptide for which the other is specific, we showed that the minimal requirement is simultaneous engagement of the TCR and class I MHC molecules of the same cell. This could occur in single cells or pairs of cells presenting peptide to each other. Thus, mechanistically the inhibition is analogous to veto, and might be called self-veto. As a clue to a possible mechanism, we found that free 1-10 peptide induced apparent downregulation of expression of specific TCR as well as interleukin 2 receptor, CD69, lymphocyte function-associated antigen 1, and CD8. This self-veto effect also has implication for in vivo immunization and mechanisms of viral escape from CTL immunity

    Semisynthesis of sperm whale myoglobin by fragment condensation.

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    A nonclassical non-Vα14Jα18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance

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    The importance of immunoregulatory T cells has become increasingly apparent. Both CD4+CD25+ T cells and CD1d-restricted NKT cells have been reported to down-regulate tumor immunity in mouse tumor models. However, the relative roles of both T cell populations have rarely been clearly distinguished in the same tumor models. In addition, CD1d-restricted NKT cells have been reported to play a critical role not only in the down-regulation of tumor immunity but also in the promotion of the immunity. However, the explanation for these apparently opposite roles in different tumor models remains unclear. We show that in four mouse tumor models in which CD1d-restricted NKT cells play a role in suppression of tumor immunity, depletion of CD4+CD25+ T cells did not induce enhancement of immunosurveillance. Surprisingly, among the two subpopulations of CD1d-restricted NKT cells, Vα14Jα18+ (type I) and Vα14Jα18− (type II) NKT cells, type I NKT cells were not necessary for the immune suppression. These unexpected results may now resolve the paradox in the role of CD1d-restricted NKT cells in the regulation of tumor immunity, in that type II NKT cells may be sufficient for negative regulation, whereas protection has been found to be mediated by α-galactosylceramide–responsive type I NKT cells

    Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

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    Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin
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