title: In vitro induction of CD8 expression on thymic pre-T cells. II. Characterization of CD3-CD4-CD8 alpha + cells generated in vitro by culturing CD25+CD3-CD4-CD8- thymocytes with T cell growth factor-beta and tumor necrosis factor-alpha.
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.
authors.affiliation: DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304.
authors.forename: T
authors.initials: T
authors.lastname: Suda
authors.forename: A
authors.initials: A
authors.lastname: Zlotnik
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D016827:CD8 Antigens / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D066298:In Vitro Techniques; D015851:Interleukin-7 / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D015375:Receptors, Interleukin-2 / Q000032:analysis; D011994:Recombinant Proteins; D016176:T-Lymphocyte Subsets / Q000166:cytology*; D013950:Thymus Gland / Q000166:cytology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000950:Antigens, Ly; D016827:CD8 Antigens; D015851:Interleukin-7; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha