Bcl-2 Expression During T-cell Development: Early Loss and Late Return Occur at Specific Stages of Commitment to Differentiation and Survival

During T-cell development CD3-CD4-CD8- (double-negative) thymocytes proliferative and produce an enormous number of CD3loCD4+CD8+ (double-positive) thymocytes which are destined to die intrathymically unless rescued by positive selection. Those which survive become mature CD3hiCD4/8+ (single-positive) cells and are the precursor of peripheral blood lymphocytes. The product of the bcl-2 protooncogene has been implicated in preventing programmed cell death and is required for prolonged lymphocyte survival following maturation. Previously we and others have reported that Bcl-2 protein expression is biphasic, being high in proliferating double-negative stem cells, low in all double-positive thymocytes except for 1-5% of these cells, and restored in mature, single-positive thymocytes. However, it remained unclear which signaling and selection events regulate Bcl-2 during T-cell maturation. Now we have utilized four-color flow cytometry in normal and genetically altered mice for a detailed analysis of Bcl-2 expression as it relates to T-cell receptor (TCR) expression and positive selection. These studies show that (i) expression of a transgenic TCR in double-negative thymocytes does not lead to premature loss of Bcl-2; thus, Bcl-2 downregulation is not solely due to TCR expression; (ii) Bcl-2 expression is lost at the early transitional CD3-/loCD4-CD8+ stage, prior to expression of CD4; (iii) the Bcl-2+ double-positive thymocytes are those which have undergone positive selection; and (iv) upregulation of Bcl-2 during positive selection requires participation of the CD4 or CD8 co-receptor. These results demonstrate that Bcl-2 and TCR expression are regulated independently during T-cell development, and suggest a role for the CD4 or CD8 co-receptor in Bcl-2 induction during positive selection.