PubMed · 4931697
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F Scheiffarth. 1971-06-04. [Kidney transplantation. Immunologic problems].. https://pubmed.ncbi.nlm.nih.gov/4931697/
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Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123(hi) and CD11c(+) subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of "new" in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction.
Triggering of the T cell receptor (TCR) leads to the production of intracellular intermediates with half-life of a few minutes. Signaling kinetics of events originating from serial TCR triggering and its relation to antigen dose was investigated. In this study we documented incremental accumulation of short-lived intermediates of the extracellular signal-regulated kinase (ERK) family, produced during successive TCR triggering. The rate and extent of the intermediate accumulation are essentially determined by the level of TCR engagement and are augmented by costimulation. ERK-1 and ERK-2 exhibit different rates of accumulation following serial receptor triggering. The data indicate that the quantitative kinetic differences in downstream signaling pathways induce qualitatively distinct biological outcomes. Although CD69, interleukin-2, and interferon-gamma (IFN-gamma) were primarily produced by high antigen doses that supported high MAPK phosphorylation, maximal interleukin-5 expression is induced by low and intermediate stimulus doses that do not support significant accumulation of activated ERK. We further demonstrated that the rate of phosphorylated ERK accumulation correlates with the duration of delay between T cell stimulation and the onset of IFN-gamma response, with stronger stimuli giving a more rapid IFN-gamma response. This delay might reflect the time required for the accumulation of signaling intermediates up to a threshold level that is necessary for activation. Thus, the data suggest that signaling events originating from serially triggered TCR are not simply sustained but are gradually accumulated and are integrated in a corresponding response.
The proteins ESAT-6 and CFP-10 have been shown to be secreted by Mycobacterium tuberculosis and Mycobacterium bovis cells, to be potent T-cell antigens, and to have a clear but as yet undefined role in tuberculosis pathogenesis. We have successfully overexpressed both ESAT-6 and CFP-10 in Escherichia coli and developed efficient purification schemes. Under in vivo-like conditions, a combination of fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy have shown that ESAT-6 contains up to 75% helical secondary structure, but little if any stable tertiary structure, and exists in a molten globule-like state. In contrast, CFP-10 was found to form an unstructured, random coil polypeptide. An exciting discovery was that ESAT-6 and CFP-10 form a tight, 1:1 complex, in which both proteins adopt a fully folded structure, with about two-thirds of the backbone in a regular helical conformation. This clearly suggests that ESAT-6 and CFP-10 are active as the complex and raises the interesting question of whether other ESAT-6/CFP-10 family proteins (22 paired genes in M. tuberculosis) also form tight, 1:1 complexes, and if so, is this limited to their genome partner, or is there scope for wider interactions within the protein family, which could provide greater functional flexibility?