Purification and Some Characteristics of Human T-Cell Growth Factor from Phytohemagglutinin-Stimulated Lymphocyte-Conditioned Media

Human T-cell growth factor (TCGF), a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration. The purified gr...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1980-10, Vol.77 (10), p.6134-6138
Main Authors: Mier, James W., Gallo, Robert C.
Format: Article
Language:English
Subjects:
Blood
Cell growth
Chromatography
Culture Media
Cultured cells
Extracellular Space - analysis
Filtration
Gels
Humans
Interleukin-2 - isolation & purification
Ion exchange
Isoelectric Point
Lectins
Lymphocytes
Lymphokines - isolation & purification
Molecular Weight
Phytohemagglutinins - pharmacology
T lymphocytes
T-Lymphocytes - analysis
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Summary:Human T-cell growth factor (TCGF), a mitogenic protein that appears in the media of cultured lymphocytes after phytohemagglutinin-stimulation, has been purified more than 400-fold from serum-free conditioned media by using a sequence of ion exchange chromatography and gel filtration. The purified growth factor elutes as a broad peak from DEAE-Sepharose, focuses diffusely at a pH of about 6.8 on isoelectric focusing (suggesting heterogeneity in electrical charge), has an estimated molecular weight of approximately 23,000 as judged by gel filtration (12,000-13,000 on NaDodSO4/polyacrylamide gel electrophoresis), is resistant to DNase and RNase, is degraded by trypsin, and does not adhere to any of several lectin-Sepharoses. These characteristics indicate that it is nonglycosylated and protein in nature. The activity of the factor, determined by cell counts or [3H]thymidine incorporation in human T lymphoblasts, is stable at room temperature in crude conditioned media, but the partially purified factor requires the addition of albumin or polyethylene glycol to maintain stability. Unlike the crude conditioned media, the purified factor lacks colony-stimulating activity and, unlike lectins, antigens, and crude conditioned media, it does not initiate blastogenesis in peripheral blood lymphocytes but is a selective mitogen for T cells that have undergone blast transformation secondary to exposure to a lectin or antigen. This indicates that the factor is a second signal in the T-cell immune response. The partially purified factor has been used to selectively grow several human T-cell lines, including cells that are cytotoxic to a variety of target cells.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.77.10.6134