Surprisingly, vitamin A-deficient mice undergoing an infection, or immunized with an antigen, had T lymphocytes that secreted IFNgamma at a 10-fold higher rate than controls. The T cells from A-deficient mice functioned poorly as stimulators of B cell antibody responses; there was a low helper T cell frequency and the cells underproduced the lymphokines IL-4 and IL-5. Repletion with retinoic acid, a vitamin A metabolite, sharply downregulated IFNgamma, restored the helper T cell frequency, and returned antibody responses to normal levels in vitro. Retinoid repletion also restored antibody responses in vivo. Cloned mouse MD13-10 T cells mimic freshly explanted immune T cells with respect to IFNgamma production. When MD 13-10 T cells are retinoid depleted and stimulated, they overproduce IFNgamma, and retinoic acid directly downregulates the IFNgamma secretion rate. We found an unexpected retinoic acid receptor- alpha (RARalpha) transcript and a novel RARalpha-immunoreactive protein in MD13-10 T cells and other T cells. Together, our findings lead us to propose as a working hypothesis that retinoic acid, acting through an undefined mechanism possibly involving this novel RARalpha isoform, specifically downregulates T cell IFNgamma production. The experiments proposed here will test our hypothesis by studying the biochemistry of retinoid-mediated IFNgamma regulation in cloned MD 13- 10 T cells. We propose to define the retinoid-sensitive IFNgamma induction pathway, to explore whether retinoid-mediated control is at the IFNgamma transcript or protein level, and to analyze the dependence of regulation on macromolecular synthesis. We propose to characterize the T cell RARalpha transcripts by Northern blotting with exon-specific probes, and sequencing of novel 5' and/or 3' cDNA ends. We will investigate the putative RARalpha proteins using immunochemistry. We plan to express and purify the novel RARalpha N-terminal fragments, and raise antibodies. We will use the antibodies to develop RARalpha isoform-specific ELISA, retinoid-binding, and DNA-binding assays and apply them to MD13-10 T cell proteins. The proposed research is significant in that it will provide new information about a regulatory relationship between two potent controllers of immune function and cell growth. IFNgamma regulates immunity, autoimmunity, hemopoiesis, viral replication, and malignant cell growth. Retinoic acid also regulates immunity, and normal and malignant cell growth and differentiation. The powerful anticancer and immunoregulatory activities of both molecules emphasize the importance of investigating how retinoic acid downregulates IFNgamma gene expression.
