Local T lymphocyte accumulation is thought to be important for tumor rejection and may presage local autoimmune disease and rejection of transplanted organs. The mechanisms of T lymphocyte accumultion at the site of initial antigen stimulation are not well defined, but it is known that antigen stimulated T cells produce a lymphokine(s) that is (are) chemotactic for other T cells. It is proposed that several different lymphokines that specifically attract either helper/inducer or suppressor/cytotoxic T cells may be released at different times and perhaps by different T lymphocyte subpopulations throughout the course of a local inflammatory response. Further, it is probable that these lymphokines play a significant role in determining the lymphocyte subpopulations that accumulate in a local inflammatory response and thereby, modulate the immune response. A T cell hybridoma, FS730.11, that produces large amounts of T cell specific lymphocyte chemotactic factor (LCF), but only a restricted range of other lymphokines has been developed. Con A stimulated supernate from cultures of FS730.11 will be used as a source of LCF from a single cell type to define whether helper or suppressor T cell subpopulations respond to this particular LCF. Helper and suppressor T cell subpopulations will be obtained by plating nylon wool non-adherent, mouse spleen cells on rat monoclonal anti-Lyt1 or Lyt2 antibody-coated plastic tissue culture plates. The purity of cell subpopulations will be assessed by flow cytrometry. Chemotaxis of these T cell subpopulations, in response to LCF, will then be assessed by migration through 8 micron pore size nitrocellulose filters in modified blindwell Boyden chambers. Semi-purified LCF will be injected into the peritoneum of mice. The peritoneal exudate cells will be quantified, differentiated by hematoxylin staining, and evaluated by flow cytometry for T lymphocyte. Purification of LCF will be initiated by sequential ultrafiltration and high performance liquid chromatography (HPLC). LCF will be characterized by sensitivity to inactivation with several enzymes, by disc gel electrophoresis, by isoelectric focusing and by column chromatography. Antibody to purified mouse LCF will be produced in rats. Intrinsically, radiolabelled LCF will be produced by Con A stimulation of FS730.11 in tissue culture, and a radioimmunoassay (RIA) for LCF developed. This RIA will facilitate future experiments to determine the T cell subpopulation(s) source of LCF and the kinetics of LCF production. A rat B cell hybridoma that secretes anti-LCF antibody will be produced, and antibody from that B cell hybridoma will be used in future experiments to study modulation of the immune response by inhibiting LCF activity in vivo.
| Bourbeau, P; Dicker, D; Higgins, M L et al. (1989) Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790. J Bacteriol 171:1982-6 |
