KINETICS OF FAS-INDUCED APOPTOSIS IN THYMIC ORGAN-CULTURE

Although most thymocytes express high levels of Fas antigen (CD95), th e role of Fas in apoptosis signaling during thymocyte maturation has n ot been defined. Fas apoptosis occurs primarily in the CD4(+)CD8(+) su bpopulations of thymocytes. Fas expression and apoptosis function were investigated in the CD4(-)8(-), CD4(+)8(+), and CD4(+) and CDS single positive thymocyte subpopulations by in vivo injection of anti-Fas an d in vitro incubation of Fas with thymic organ cultures. Fas was first expressed on CD4(-)8(-) thymocytes coincident with expression of IL-2 R and CD44. In Fas mutant lpr/lpr mice, defective Fas expression corre lated with overproduction of late-stage CD4(-)8(-)-thymocytes. Fas was highly expressed on CD3(dull) and CD3(bright) thymocytes. CD4(+)8(+)C D3(dull) thymocytes were sensitive to Fas apoptosis, whereas more matu re CD4(+)8(+)CD3(bright) thymocytes were resistant to Fas apoptosis. A nti-Fas incubation with established thymic organ culture for 24 hr res ulted in apoptosis of approximately 25% of thymocytes. Continued incub ation of thymic organ culture with anti-Fas resulted in an apoptosis r ate of 25% of CD4(+)CD8(+) thymocytes per day for the first 3 days of culture. Continued culture for further time points up to 6 days did no t result in further apoptosis of the CD4(+)CD8(+) thymocytes. These re sults suggest that CD4(-)CD8(-)CD44(+) IL-2R(+) thymocytes express Fas and there is overpopulation of the subsequent developmental stage of thymocytes in Fas mutant lpr mice. Also, early-stage CD4(+)8(+) thymoc ytes are susceptible to Fas apoptosis, whereas Fas apoptosis resistanc e is required after 3 days of thymic organ culture. We conclude that t hese two populations of thymocytes me susceptible to Fas ligand-mediat ed apoptosis during T cell development in the thymus.