Development of T cell receptor alpha beta-bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration.

Organ culture (OC) of murine fetal thymuses on a membrane filter floating on the medium has been used as an important strategy in studies of the mechanism of T cell development. On the other hand, the submersion organ culture (S-OC) system, in which fetal thymus lobes are submerged in the culture medium, is not popularly used because the growth of T cells is much lower than that in OC at the air-medium border (AMB-OC). In the present work, we tried to culture the fetal thymuses in the S-OC system at 5% CO2 and various concentrations of O2. We found that in the environment containing 5% O2, gamma delta T cells were selectively generated, though the cell recovery was less than one-tenth of that seen in AMB-OC. Generation of gamma delta T cells was hardly affected by increasing the O2 concentration. In marked contrast, the development of alpha beta T cells was highly dependent upon the O2 concentration. In the S-OC at more than 60% O2, differentiation and growth of T cells, as determined in terms of recovered cell number, CD4 vs. CD8 profile and predominance of alpha beta lineage, were comparable to those observed in AMB-OC. It was further shown that clonal deletion of V beta 6+ T cells occurred in high O2 S-OC of CBA/J (Mls-1a) but not C3H/HeJ (Mls-1b) mice, the extent of the deletion being low but comparable with that seen in AMB-OC. These results indicated that the fetal thymus microenvironment was potentially capable of supporting the development of both gamma delta and alpha beta T cells, and that skewing to one of these lineages was determined by an additional factor(s) such as the concentration of O2 dissolved in the medium.