Isolation of proliferation factor of immature T-cell clone in concanavalin A-stimulated splenocyte culture supernatant

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.

Figure 1

Purification of NPF. (a) Asahipak GS-320 HPLC of active Sep-pak C18 fraction using PBS as a solvent. (b) Cosmosil C18 HPLC of active Asahipak GS-320 fraction. The elution was done with a linear gradient of increasing concentrations of 2-PrOH/AcCN (7 : 3) in 0·05% TFA. An aliquot of each fraction was subjected to the proliferation assay of N-9F by an MTT method.

Figure 2

SDS–PAGE of NPF. NPF was subjected to electrophoresis on a 17% gel and detected by silver staining. (a) Protein MW standard; lysozyme (50 ng) (b), (25 ng) (c), (12·5 ng) (d); and (e) NPF.

Figure 3

N-9F-Proliferation activity of NPF. (a) N-9F cells were cultured with NPF in RPMI-1640 medium, and proliferation of N-9F was measured by an MTT method. N-9F cells were cultured with NPF in the presence or absence of hamster anti-NPF antiserum (2%), and proliferation of N-9F was measured by an incorporation of [³H]thymidine. The mean background is 19 256 c.p.m. (b) Immature thymocytes (E17), adult thymocytes and adult splenocytes were cultured with NPF in serum-free RPMI-1640 medium, and cell proliferation was measured by [³H]thymidine incorporation. Mean backgrounds are 565 c.p.m. for immature thymocytes, 492 c.p.m. for adult thymocytes and 4158 c.p.m. for adult splenocytes, respectively. Data represent mean from three to five independent experiments. SD values are shown as error bars.

Figure 4

Effect of NPF on cell cycle of N-9F cells. N-9F cells were cultured with sample in RPMI-1640–10% FCS (RPMI(+)) or serum-free RPMI-1640 (RPMI(–)). DNA content of cells were determined by flow cytometric analysis. (a) Representative flow cytometric analysis. The results from three independent experiments by NPF (2·4 ng/ml) are shown in (b) and (c). Total cell numbers ( × 10⁵) were 0·42 ± 0·27 for medium and 1·09 ± 0·13 (P < 0·05 versus medium) for NPF in RPMI(+), and 0·35 ± 0·17 for medium and 0·28 ± 0·11 for NPF in RPMI(–), respectively. Data represent the mean ± SD. Statistically significant from medium: *P < 0·05, **P < 0·01.

Figure 5

Effect of NPF on FTOC. Thymus lobes from fetal day 14 mice were cultured with NPF or antiserum and stained with anti-CD4 and anti-CD8 antibodies. Each of thymocytes in five lobes and all thymocytes emigrated on membrane from five lobes were subjected to flow cytometric analysis. (a) Representative flow cytometric analysis by NPF for 6 days. (b) Representative flow cytometric analysis by hamster anti-NPF antiserum (2%) for 4 days. (c) The results from five lobes by NPF (2·4 ng/ml) are shown. Five independent experiments for thymus lobes and two independent experiments for thymocytes emigrated on membrane were performed with similar results. Total cell numbers ( × 10⁵) per thymus lobe were 2·06 ± 0·50 for medium and 4·04 ± 0·74 (P < 0·05 versus medium) for NPF in thymus lobes, and 1·08 for medium and 1·16 for NPF in thymocytes emigrated on membrane, respectively. (d) The results from 5 lobes by hamster anti-NPF antiserum (2%) are shown. Total cell numbers ( × 10⁵) per thymus lobe were 2·60 ± 0·65 for medium and 2·45 ± 0·43 for NPF, respectively. Data represent the mean ± SD, except thymocytes emigrated on membrane. Statistically significant from medium or normal serum: *P < 0·05, **P < 0·01.

Figure 6

Western blotting of TCGF with rabbit anti-mrH-saposin A antibody. Serum-free TCGF concentrated 10-fold was used as TCGF. αOVA: rabbit anti-ovalbumin immunoglobulin G, αNPF: rabbit anti-mrH-saposin A immunoglobulin G.