The goal of this project is to broaden our understanding of the physiology and regulation of early events in T cell activation. Lymphocyte metabolism and effector function expression are regulated by antigen/mitogen and lymphokine binding to cell surface receptors. We are investigating consequences of mitogen mediated signals by isolating and characterizing genes which are transcriptionally regulated by these events. We expect that genes induced within a few hours after antigen or mitogen activation of lymphocytes will encode functions that are fundamentally important for the initiation of proliferation and effector function expression in these cells. Known induced early genes include oncogenes (c-myc and c-fos), lymphokines (IL-2, gamma-IFN, GM- CSF), and lymphokine receptors (IL-2 receptor), all of which are thought to have significant effects on T cell proliferation and effector function. We have constructed a subtracted cDNA library enriched for genes that are transcriptionally induced within four hours after stimulating peripheral blood T cells with PHA and PMA. After amplification, 20,000 phage were screened with a subtracted cDNA probe enriched for mitogen-induced mRNA's, and 528 positively hybridizing clones were isolated. Cross-hybridization studies show that greater than forty unique genes have been isolated. Although known induced genes (c-myc and IL-2 receptor) are included within these clones as expected, the large majority of clones represent novel, as yet undescribed genes. We have begun characterizing inducible gene sequences with regard to 1) structure, i.e. sequencing analyses, 2) expression pattern analyses that allow broad categorization of potential function, and 3) expression pattern analyses that define distinct patterns of gene regulation. Initial results indicate that a variety of classes have been isolated including T cell-specific, lymphocyte-specific, and proliferation-specific genes. In addition, distinct regulatory networks acting upon these induced genes have been defined by differential kinetics of expression and discriminating effects of the drug cyclosporin A and the HTLV-I encoded trans-activating factor (TATI) upon transcription patterns.
