The T cell colony assay is a valuable technique for assessing factors controlling normal human lymphopoiesis. In this procedure, mononuclear cells or isolated T lymphocytes are plated in semi-solid media and growth is stimulated by a non-specific mitogen, usually PHA. Previously, we have shown that the clonal expansion of PHA-stimulated isolated T cells depends on the presence of a soluble initiating factor. Both IL-1 and IL-2 will subserve this function. In addition, initiating factors produced by tumor cell lines of both B lymphocytic and monocytic lineages will promote colony growth. Of note, the factors from these tumors appear to be different than the classic interleukins. The proposed studies are designed to further define the cellular and soluble factor requirements for clonal growth.
The specific aims i nclude: (1) biochemical characterization and purification of the soluble factor elaborated by Raji cells (a B lymphoma line), a potent initiator of colony responses, and supernatants obtained from other cell lines (2) determinations of the T cell subsets, separated by monoclonal antibody techniques, capable of forming colonies (3) evaluations of the potential regulatory influences of specific subsets of mononuclear cells and soluble products derived from these cells on the T cell growth, (4) examinations of the activities of immunosuppressive drugs on clonal responses (5) measurements of the functional activities of cells contained in colonies by expanding them in IL-2 containing media and determining their activities in the pokeweed-induced immunoglobulin synthesis assay and (6) comparisons of the mitogenic activities of phorbol myristate acetate (TPA) and Sepharose-bound T3 with PHA. These compounds appear to activate T cells by mechanisms distinct from those expressed by PHA. The second major aspect of this study is to evaluate clonal responses associated with several disorders. A principal component of these studies is to further evaluate the suppressed colony responses seen in patients with all clinical phases of HTLV-III infections. Initial results suggest that several factors may be responsible for the growth defects seen in this syndrome; these include decreased productions of IL-2, deficits in the ability of cells to express IL-2 receptors and the presence of various inhibitory factors. The proposed studies will define the mechanisms responsible for this growth abnormality. In addition, studies aimed at determining the prognostic value of the colony assay will be performed.
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