This application is based on two new paradigms to explain two enigmas regarding the function of Fas. First, in the absence of Fas expression in lpr mice, a phenotypically unusual population of CD4-8- B220+ TCR alpha beta+ cells accumulates. Paradigm 1 proposes that this phenotype results from high intensity TCR signals which, in normal mice, results in apoptosis. Second, Fas (like CD28) activates Jun kinase (JNK) and can induce apoptosis, as well as costimulate with CD3 on resting T cells to augment IL-2 production and proliferation. Paradigm 2 suggests that the balance of MAPK and JNK determines the functional outcome; dominance of JNK will favor apoptosis, whereas a balance of MAPK/JNK rescues cells and favors proliferation. Specific Aim 1 develops Paradigm 1 by use of a new TCR transgenic lpr strain known as OVA-tcr-1 lpr/lpr that recognizes ovalbumin (OVA) peptide, SIINFEKL. Administration of SIINFEKL variants will confer different signal intensities. Moderate TCR signals will yield accumulation of only CD8+ cells, while peptides providing a high intensity signal will promote CD4-8- clonotype TCR+ cells. The latter will be deleted (detected by TUNEL assay) in OVA-tcr-1 +/+ mice and retained in OVA-tcr-1 lpr/lpr mice. The second part will address another long standing debate whether lpr CD4-8- T cells are generated in the thymus or periphery. Thymectomy of beta 2m-/- lpr/lpr mice and beta 2m+/+ lpr/lpr mice, will be followed by thymic transplant of the opposite genotype. This will selectively re-express class I in either the thymus or the periphery, and appearance of CD4-8- T cells monitored. Specific Aim 2 develops Paradigm 2 by examining the importance of JNK activation in Fas signaling using dominant positive and negative variants of downstream c-Jun, and upstream Rac and Cdc42, members of the Rho family of GTP-binding proteins that activate JNK. In addition, rescue from apoptosis by Fas will be attempted using a dominant positive MAPK. Finally, this model is applied to two normal immune situations of apoptosis versus proliferation to examine the levels of MAPK and JNK. Specific Aim 3 extends Paradigm 2 to Fas costimulation with CD3 of IL-2. The prediction is that Fas will enhance activity of transcription factors that use c-Jun, namely AP-1 and NFAT. Functional confirmation will use NFAT-luciferase and AP-1-luciferase transgenic mice. Finally, a novel alternate lpr NFAT-binding suppressor protein will be purified, and its suppressor function confirmed using NFAT-luciferase/lpr mice.
NIH ExPorter
NIH ExPorter