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2. Concanavalin A stimulation of mouse lymphocytes at low concentration II. THE EFFECT OF CONDITIONED MEDIUM FROM PERITONEAL EXUDATE CELLS AND FROM LYMPHOCYTE CULTURES.
- Author
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Young, Barbara
- Subjects
- *
LYMPHOCYTES , *SPLEEN , *EXUDATES & transudates , *CELLS , *CELL culture , *BIOLOGICAL assay , *T cells ,MICE anatomy - Abstract
The first paper in this series reported that it was possible to reconstitute the response of low concentrations of mouse spleen lymphocytes to concanavalin A (Con A) by adding small numbers of peritoneal exudate cells (PEC) to the cultures. In this paper it is shown that the role of the PEC in this system can be partially replaced by conditioned medium (CM) prepared from PEC cultures or completely replaced by CM taken from lymphocytes cultured at optimal concentration. These CM were inactive unless fresh Con A was added to the assay cultures. Activity was present in CM which was incubated with lymphocytes or PEC for the shortest possible time but maximal activity was found after 24 hr of incubation. Activity was also found in CM prepared in the absence of Con A. Only in the case of lymphocytes cultured at optimal concentration for 24 hr was there substantially more activity in the CM thus prepared if Con A was present. PEC preparations depleted of T lymphocytes produced as much activity in CM as the untreated control. CM produced by PEC was less sensitive to heat treatment or to freezing and thawing than that produced by lymphocyte cultures. [ABSTRACT FROM AUTHOR]
- Published
- 1982
3. The method of deriving human T-cell clones alters the proportion of IL-10-producing cells.
- Author
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Cohen, S. B. A., Webb, L. M. C., and Feldmann, M.
- Subjects
- *
IMMUNOLOGY , *CLONE cells , *INTERLEUKIN-10 , *BLOOD cells , *T cells , *CYTOKINES , *MITOGENS , *CELL culture - Abstract
It is now well documented that the mode of primary stimulation in the mouse determines the ratio of Th1-, Th0- or Th2-type T cells obtained. In this paper we determine whether driving and cloning human peripheral blood mononuclear cells (PBMC) non-specifically with interleukin-2 (IL-2) and/ or IL-4 and mitogen, will differentially select T cells for IL-10 production. The presence of IL-2 during culture (with or without IL-4) yielded predominantly Th1-type clones (81% of clones with IL-2 alone and 77% with IL-2 + IL-4) and a low frequency of Th0-type clones (19% of clones with IL-2 and 10% of clones with IL-2 + IL-4), whereas IL-4 alone increased the yield of the IL-4 producing Th0 (35%) clones. The proportions of IL-2-producing clones did not alter with treatment; however, the combination of IL-2 + IL-4 altered the proportions of IL-10-producing clones. 47% of IL-2-cultured cells and 51% of IL-4-cultured cells produced IL-10 respectively, whereas only 22% of clones driven with IL-2 + IL-4 produced detectable levels of IL-10. Thus in the preliminary experiments described here we have demonstrated that the cytokines used to initially drive out human T cells and maintain the T-cell growth can skew the Th1/Th2/Th0 cytokine profiles of the clones obtained from mitogen-stimulated cultures. Moreover and unexpectedly the proportion of IL-10-producing cells is altered. These results are important in interpreting analysis of the cytokine profiles of human T cells and raise questions concerning how we should derive T-cell clones for a cytokine analysis that truly reflects the in vivo situation. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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4. H-2-associated effects of flanking residues on the recognition of a permissive mycobacterial T-cell epitope.
- Author
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Roman, E., Harris, D. P., Jurcevic, S., Ivanyi, J., and Moreno, C.
- Subjects
- *
MYCOBACTERIUM tuberculosis , *MAJOR histocompatibility complex , *T cells , *CELL culture , *TUBERCULOSIS , *TUBERCULIN - Abstract
Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19 000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype. [ABSTRACT FROM AUTHOR]
- Published
- 1995
5. Short-term lymphokine stimulation of human peripheral blood mononuclear cells generates cytolytic activity against endothelial cells: involvement of natural killer cells.
- Author
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Miltenburg, A. M. M., Meijer-Paape, M. E., Daha, M. R., and Paul, L. C.
- Subjects
- *
INTERLEUKIN-2 , *T cells , *MONONUCLEOSIS , *CELL culture , *HYDROBROMIC acid , *MONOCLONAL antibodies - Abstract
We previously reported that incubation of human peripheral blood mononuclear cells (PBMC) for 5 days with T-cell growth factor (ICGF) resulted in lymphokine-activated killer activity against endothelial cells (EC). In this paper we report on the effects of short-term incubation of PBMC with lymphokines. We show that incubation of PBMC with lymphokines during an 18-hr period is sufficient to generate a strong cytolytic response against EC. The cytolytic capacity of the effector cells was directly dependent on the dose of lymphokine added during the induction phase. When PBMC were separated into adherent and non-adherent cells, the non-adherent fraction could be induced to lytic activity against EC, whereas the adherent cells could not. When PBMC were separated, using 2-amino-ethylisothiouronium bromide hydrobromide-treated sheep red blood cells (AET-SRBC), into T- and non-T-cell fractions, the latter fraction could be induced to lyse EC. TCGF-induced cell-mediated EC lysis could not be inhibited using anti-T3 nor antie-LFA-l antibodies. Lysis of EC by TCGF-stimulated effector cells was strongly inhibited by the addition of unlabelled K562 target cells, whereas cold OKT3 hybridoma cells did not exert such an effect. In conclusion: the kinetics of the induction of lytic activity against EC, as well as the cell separation experiments, suggest that short-term-activated NK cells may lyse EC. This hypothesis was confirmed using monoclonal antibody and cold target cell analysis. [ABSTRACT FROM AUTHOR]
- Published
- 1988
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