Discovery of Immature Thymocyte Proliferation Factor

Abstract

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 N-9F at 10 -12  10 -9 M 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, single positive and double positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF neutralized the increasing eŠect of NPF on immature thymocytes. All eŠects of NPF was inhibited by mAb QR6.6 to recognize a 100kDa surface molecule of N-9F. The amino-terminal 20 amino acid sequence of NPF was identiˆed and identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A Hitrap-mouse recombinant His-tag-saposin A antibody column bound NPF, pulled down the NPF activity in TCGF, and the antibody recognized a 16kDa molecule in western-blotting of TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and diŠerentiation of immature thymocytes. The physiological signiˆcance of NPF in the growth and diŠerentia-tion of immature thymocytes was discussed in view of the characteristic distributions of NPF and the molecule recognized by its mAb QR6.6 in fetal thymi.