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Isolation of proliferation factor of immature T‐cell clone in concanavalin A‐stimulated splenocyte culture supernatant
Author(s) -
Kohama Yasuhiro,
Shinoda Shinji,
Hagihara Katsunobu,
Hashimoto Takeshi,
Yamaguchi Asuka,
Nakamura Akihiro,
Tsuchiya Takahiro,
Tsujikawa Kazutake,
Yamamoto Hiroshi
Publication year - 2003
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1046/j.1365-2567.2003.01642.x
Subject(s) - splenocyte , concanavalin a , biology , microbiology and biotechnology , antiserum , cd8 , clone (java method) , thymocyte , cell growth , antibody , biochemistry , dna , antigen , immunology , in vitro
Summary An athymic mouse‐derived immature T‐cell clone, N‐9F, was not maintained by interleukin‐2 alone but required another soluble factor, contained in concanavalin A‐stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N‐9F‐proliferation factor (NPF) was isolated in a pure form from TCGF. N‐9F cells and immature thymocytes proliferated in the presence of NPF at 10 −11 −10 −8 g/ml in a dose‐dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N‐9F. NPF increased CD4 and CD8 double negative thymocytes and CD8 single positive thymocytes in fetal thymus organ culture. A hamster anti‐NPF antiserum possessing the capacity to neutralize N‐9F proliferation activity of NPF decreased double negative thymocytes. The amino‐terminal amino acid sequence of NPF was identified to be Ser‐Leu‐Pro‐Cys‐Asp‐Ile‐Cys‐Lys‐Thr‐Val‐Val‐Thr‐Glu‐Ala‐Cys‐Asn‐Leu‐Leu‐Lys‐Asp‐ and was identical to that of rat saposin A. The apparent molecular weight of NPF, 16 000, was comparable to that of saposin A. A rabbit anti‐mouse recombinant His‐tag (mrH)‐saposin A antibody recognized a 16 000 MW molecule in TCGF. A Hitrap‐saposin A antibody column bound NPF and pulled down the NPF activity in TCGF. Thus, NPF in TCGF was a saposin A‐like protein possessing the capacity for growth and differentiation of immature thymocytes.