Recently we have developed an assay for cytokines needed for the in vitro generation of primary murine cytolytic T lymphocytes (CTL) against alloantigens on tumor cells. The factors which function in the assay are called CHF for CTL helper factor. This assay can detect previously uncharacterized cytokines including a factor of 30,000 molecular weight (RDCHF) made by the cell line RDM4. We have been able to concentrate this cytokine by adsorption to Sepralyte and to obtain a good degree of purification using reverse phase HPLC. We will continue to purify this molecule to a single major protein species for use in biological experiments and for obtaining an N terminal amino acid sequence. We will confirm that the protein we begin to sequence is actually RDCHF by cloning the molecule using recombinant DNA techniques. We have already accomplished the key step in this procedure, the expression of the molecule in Xenopus laeis oocytes. Thus we are able to assay the mRNA specific for the cytokine. Our next steps are: (1) Construction of cDNA libraries. (2) Identification of RDCHF cDNA. (3) Analysis of the nucleotide sequence of RDCHF cDNA. (4) Expression of recombinant RDCHF cDNA in mammalian cells. Once we have obtained reasonably homogeneous RDCHF, we will test it for its biochemical properties. We will also test its biological properties including the binding of radiolabeled RDCHF to different cells involved in CTL generation, the addition of RDCHF to spleen cells depleted of certain cell types, and the function of RDCHF in other help deficient systems for the generation of CTL in vitro. We have found novel molecules with CHF activity from not only RDM4 but also other cell lines such as EL4 and ASFTL-1. We propose to repeat the studies described above with these cytokines.
| Fan, D P (1993) Quantitative estimates for the effects of AIDS public education on HIV infections. Int J Biomed Comput 33:157-77 |
