Genes encoding TNF, LTalpha and LTbeta, (the two components of the membrane-bound lymphotoxin) are closely linked at the telomeric border of the class II region of the MHC, and they show different pattern of regulation of expression. We are dissecting regulatory mechanisms controlling TNF inducible expression in macrophages/monocytes. We have identified two components in transcriptional activation of TNF gene by LPS: i.e. initiation and elongation (processivity) and we have determined sequence requirements for activation of the human TNF promoter by LPS. We have continued our genetic studies to address the possibility that predisposition to autoimmune and infectious disease may be linked to variation at the TNF/LT locus. Although we detect linkage of TNF genotypes to IDDM and sepsis (which may be secondary, due to linkage disequilibrium in the MHC), we are unable to correlate promoter activity with natural sequence variations in the TNF promoter (such as -308 polymorphism known to be linked to predisposition to cerebral malaria). At present we are determining requirements for TNF activation by malaria toxin. We have cloned and characterized the murine LT gene and determined its pattern of expression in vitro and in situ. We have shown that cooperation between transcriptional factors of three families is essential for inducible transcription of this gene in a T cell line. Finally, as a direct approach to assess the biological function of the cytokines of the TNF/LT subfamily, we have initiated an international collaboration aimed at generating strains of mice with targeted deletions of TNF/LT genes in conventional as well as in inducible fashion. During last year we have obtained and initially characterized mice with double inactivation of LTalpha/LTbeta genes and with deletion of the entire TNF/LT locus (triple knock-out.
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