Development of T cell receptor αβ‐bearing T cells in the submersion organ culture of murine fetal thymus at high oxygen concentration

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Abstract
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, γδ cells were selectively generated, though the cell recovery was less than one‐tenth of that seen in AMB‐OC. Generation of γδ T cells was hardly affected by increasing the O2 concentration. In marked contrast, the development of αβ 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 αβ lineage, were comparable to those observed in AMB‐OC. It was further shown that clonal deletion of Vβ6+ 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 γδ and αβ 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.