Abstract
We previously reported that CD25 (IL-2R p55)-positive CD3-CD4-CD8- murine thymocytes can be induced to express CD8 alpha (Lyt-2) by transforming growth factor-beta plus TNF-alpha in the presence of IL-7 (which is necessary to maintain the viability and differentiation capacity of CD25+CD3-CD4-CD8- thymocytes in vitro). The majority of cells recovered after 2 to 3 days from these cultures expressed CD8 alpha (but not CD3 or CD4). In this study, we have characterized these in vitro generated CD3-CD4-CD8 alpha + thymocytes and compared them with normal CD3-CD4-CD8+ thymocytes. Unlike normal CD3-CD4-CD8+ thymocytes that express CD8 alpha and CD8 beta (Lyt-3-chain) simultaneously, only a fraction of in vitro generated CD3-CD4-CD8 alpha + cells expressed CD8 beta. However, along with the induction of CD8 alpha and CD8 beta expression, the expression of other T cell differentiation markers (including CD2, CD25, and CD44) also changed in a manner corresponding to physiologic differentiation. Cell-surface phenotyping suggests that CD8 alpha + beta - cells are less mature than CD8 alpha + beta + cells. These in vitro generated CD3-CD4-CD8 alpha + thymocytes expanded and differentiated into the CD4+CD8+ stage as well as mature (CD3+) single positive (CD4+CD8-) and CD4-CD8+) stages in fetal thymus organ culture that had been depleted of lymphoid cells by treatment with 2-deoxyguanosine. The latter observation indicates that these in vitro generated CD3-CD4-CD8 alpha + thymocytes are responsive to other differentiation-inducing signals (including those that induce CD4) that exist in fetal thymus organ culture. These results suggest that in vitro generated CD3-CD4-CD8 alpha + thymocytes represent intermediate differentiation stages between CD25+CD3-CD4-CD8- and CD3-CD4-CD8+ cells found in normal thymus.
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