Cellular and Peptide Requirements for in vitro Clonal Deletion of Immature Thymocytes

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 19; pp. 9000 - 9004
• Main Authors: Iwabuchi, Kazuya, Nakayama, Kei-Ichi, McCoy, Roderick L., Wang, Fanping, Nishimura, Takashi, Habu, Sonoko, Murphy, Kenneth M., Loh, Dennis Y.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.10.1992; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; Analysis of the immune response. Humoral and cellular immunity; Animals; Antigen-Presenting Cells - immunology; Apoptosis; B lymphocytes; B-Lymphocytes - immunology; Biological and medical sciences; CD4 Antigens - immunology; CD8 Antigens - immunology; Cell Line; Cell lines; Cells, Cultured; Cellular biology; Chickens; Clonal deletion; Crosses, Genetic; DNA - genetics; DNA - isolation & purification; Epithelial cells; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Genes, MHC Class II; Immunobiology; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Transgenic; Molecular Sequence Data; Organs and cells involved in the immune response; Ovalbumin - immunology; Peptides - chemical synthesis; Peptides - immunology; Peptides - pharmacology; Proteins; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - immunology; Rodents; Spleen cells; T cell antigen receptors; T lymphocytes; T-Lymphocytes - cytology; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; Thymocytes; Thymus Gland - cytology; Thymus Gland - immunology; Transgenic animals; Nurse cell; Ovalbumin; T cell receptor; Thymocyte; Rodentia; Transgenic animal; Glycoproteins; In vitro; Precursor; Vertebrata; Mammalia; Depletion; Cell line; Mouse; Cell death; Clone
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.19.9000

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Adon their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration <10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus.