A CD4+ T cell line-secreted factor, growth promoting for normal and leukemic B cells, identified as thioredoxin

• Published in: International immunology 1995-04, Vol.7 (4), p.625-633
• Main Authors: Rosén, Anders, Lundman, Pia, Carlsson, Mats, Bhavani, Kasibhatla, Srinivasa, Bachally R., Kjellström, Gunilla, Nilsson, Kenneth, Holmgren, Arne
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies; B-Lymphocytes - drug effects; B-Lymphocytes - physiology; B-type chronic lymphocytic leukemia; CD4-Positive T-Lymphocytes - metabolism; Cell Line; Cell-Free System; Chromatography, Affinity; Clone Cells; Culture Media, Conditioned - analysis; cytokine; Cytokines - analysis; Drug Synergism; Growth Substances - biosynthesis; Growth Substances - isolation & purification; Growth Substances - pharmacology; Humans; IL-2; IL-4; IL-6; Leukemia, Lymphocytic, Chronic, B-Cell - metabolism; Medicin och hälsovetenskap; Molecular Sequence Data; Radioimmunoassay; redox regulation; thioredoxin; Thioredoxins - immunology; Thioredoxins - isolation & purification; tumor necrosis factor-α
• ISSN: 0953-8178; 1460-2377
• DOI: 10.1093/intimm/7.4.625

In this study, a B cell growth stimulatory factor, constitutlvely secreted by a human CD4+ T cell hybrldoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltratlon and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymldine incorporation In normal and leukemic (B-CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithlothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30–50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin dlsulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified ˜100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was Identical to the previously published sequence of human Trx from HTLV-I transformed T cells, adult T cell leukemia-derived factor/Trx. A minor fraction (˜30%) of the purified Trx showed alternative amIno acids at eight positions; the relevance of which is discussed. MP6-derived Trx showed prominent growth stimulatory activity, measured as [3H]thymldine incorporation, and synergy was detected, particularly with IL-2, but also with IL-1β, IL-4, IL-6, tumor necrosis factor-α, IFN-γ, low molecular weight B cell growth factor and antl-CD40 mAbs. Interestingly, the promotor for the trx gene was recently reported to contain several sequence motives compatible with regulated inducible transcription, especially by cytokines. Together with our own previous results showing a cytokine/mitogen inducible autocrine secretion of Trx from B cells, our findings point to a crucial role for extracellular Trx In redox-controlled mechanisms of the B lymphocyte activation cascade.