Pretreatment of mouse L cells with interferon

No abstract

Continuous cell lines could be reproducibly established by culturing spleen cells from adult mice injected with MLV-producer cells or directly infected with Mo-MLV with rIL-2, whereas the culture of normal splenic cells with rIL-2 induced only transient and limited proliferation resulting in no such lines. All of the lines showed morphological characteristics as LGL with Thy-1+,Lyt-1-,L3T4-,Lyt-2-,AsGM1+,FcR gamma+ phenotype without exception, and most of them exhibited typical NK-patterned cytotoxicity. Analysis of reverse transcriptase activity of the culture supernatants as well as Southern hybridization of the DNA from the lines using an Mo-MLV-specific cDNA probe indicated no evidence of retroviral replication or proviral integration, suggesting that the generation of cell lines reflected a reactive process and viral infection was not directly responsible. It was subsequently revealed that Thy-1+,Lyt-1+,Lyt-2- spleen cells from mice infected with Mo-MLV in vivo spontaneously produced surprising amounts of IL-3 in vitro, leading to the possibility that IL-3 was responsible for the generation of lines. The possibility was directly supported by the observation that continuous lines with identical characteristics could be generated completely in vitro by sequential stimulation with rIL-3 and rIL-2 from normal spleen cells without any involvement of Mo-MLV. The C beta gene of TCR was shown to be rearranged in all the lines examined, indicating the LGL lines were all genetically committed to T cell lineage. Unlike the situation in normal splenic populations expanded by rIL-2, where the expression of IL-2-R was progressively lost, constitutive expression of high-affinity-IL-2-R was observed in all the lines and thus, this was considered to explain the unlimited proliferation of them in response to rIL-2 alone. These results suggested the probable role of IL-3 in the regulation of growth and differentiation of a set of LGL committed to T cell lineage. The possible implications of the phenomenon in the regulation of hematopoiesis as well as in the control of Mo-MLV-induced leukemogenesis were discussed.

No abstract

A new procedure of assaying interferon (IF) has been developed. Cell suspension was dispensed into liquid scintillation counting vials together with IF sample. During an overnight incubation, the cells adhered sufficiently to the bottom of the vials and all the subsequent procedures were carried out without transfer of the cells from the vials. Vesicular stomatitis virus was inoculated and virusspecific RNA was labeled by adding 3H-uridine and actinomycin D to the medium. Incubation was terminated prior to completion of a single-step growth of the virus and radioactivity of the labeled cells in each vial was determined.The reciprocal of the IF dilution which reduced the radioactivity in viral RNA by 50% was taken as the titer. The present procedure consists of simple manipulations and can be completed within 24 hr. Furthermore, it is quite reproducible and gives a titer almost identical to that obtained by the conventional plaque-reduction dose method. The procedure can be applied to mouse L cells, rabbit RK-13 cells and human FL cells, without modification.The technique which is currently used most widely for assaying interferon (IF) is the plaque-reduction dose method (PRD50 method). The procedure gives reliable results but involves a longer time and laborious handlings. In order to overcome these disadvantages, several groups of workers have introduced techniques based on inhibition of nucleic acid synthesis (INAS50 method) [1,4 6], in which the IF titer is determined by measuring radioactivity of 3H-uridine incorporated into virus-specific RNA in the IFtreated cells. Although their methods have some advantages over the PRD50 method, these are still laborious and remain impractical as a routine method. In this communication we describe a new procedure of the INAS50 method which yields reliable results much faster in a simpler way. This technique will be well qualified to replace the PRD50 method. MATERIALS AND METHODSCells and media. Mouse L cells, human FL cells and rabbit RK-13 cells were employed for assaying IF preparations of the corresponding origin. Two different lines of mouse L cells (L-MS cells) and human FL cells (FL-MS cells) had been adapted to grow readily either in monolayer or in suspension in our laboratory [9]. The growth medium employed for monolayer culture was Eagle's minimum essential medium (Nissui Seiyaku Co., Japan) plus 5% calf serum (MEM + 5% CS). For suspension culture Eagle's spinner culture medium (Nissui Seiyaku Co., Japan) supplemented with 5% calf serum (SM+5%CS) was used.