Isolation of Proliferation Factor of Immature T-cell Clone in Concanavalin A-stimulated Splenocyte Culture Supernatant

Overview

Journal Immunology

Specialty Allergy & Immunology

Date 2003 May 22

PMID 12757615

Authors

Yasuhiro Kohama

Shinji Shinoda

Katsunobu Hagihara

Takeshi Hashimoto

Asuka Yamaguchi

Akihiro Nakamura

Takahiro Tsuchiya

Kazutake Tsujikawa

Hiroshi Yamamoto

Affiliations

Soon will be listed here.

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 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, 16000, was comparable to that of saposin A. A rabbit anti-mouse recombinant His-tag (mrH)-saposin A antibody recognized a 16000 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.

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