The role of antigen presenting cells (APC) is the activation of human T cells will be studied in culture. We will emphasize a) dendritic cells (DC) and monocytes as APC b) primary populations of CD4+ lymphocytes as responders and c) surface molecules and interleukins that medidate the APC-T interaction. The first set of molecules to be studied are the surface constituents of monocytes and DC. DC will be enriched with new methods that do not require antibody and complement mediated lysis, so that they can be analyzed on the FACS with MAb to Fc receptors, the LPA-1 family, and HLA class II antigens. A major aim is to propare MAb using DC from arthritis effusions as immunogen. The second set of products are cytokines. Bioassays, polyclonal AB, and cDNA probes are available to follow several cytokines in the culture medium and at the single cell level. Current probes are to IL-1, TNF, interferons, and a new candidate chemotactic factor gamma-IP-10. Blood and inflammatory monocytes and DC will be compared following stimulation with such agents as LPS, PMA, IFN-gamma, poly IC. The third set of molecules are those which underlie the APC-T cell interaction in discrete cell aggregates. Aggregates are induced by DC in the MLR and are the site for antigen-specific T cell activation. a) Production of, and responsiveness to, activating factors like IL1 will be evaluated using T cells that have just clustered with APC. b) We will try to grow clustered antigen-specific T cells in bulk and cloning cultures in the presence of IL2, DC, irradiated clusters, or their conditioned medium. c) The surface molecules that are required for antigen- dependent and independent binding of T blasts to APC will be quantitated in rapid binding assays. A panel of MAb will be tested for blocking and destabilizing effgects on DC-T clusters, e.g., LFA-1, T4, gp 44 and T11. The information from the MLR will then be extended to two other models: polyclonal (con A, OKT3) mitogenesis in which monocytes cluster with T cells, and memory helper cells which can be activated 1-2 days more rapidly than unprimed lymphocytes. In each system, the capacity of T cells to cluster APC and to become responsive to single stimuli like IL-1, PMA, and OKT3 MAb will be analyzed. The work should clarify the functions of existing cytokines and surface antigens, and identify new molecules on DC and T cells that mediate immune responses in man.
