Having identified a regulatory feedback loop in adult mice that coordinates the cyclical exportation of prothymocytes from the bone marrow with their gated importation by the thymus, we now plan to define the underlying mechanisms for this thymus-BM feedback loop. By analogy with the fetal thymus, we postulate that the cyclical generation and mobilization of prothymocytes in the adult mouse is due to the periodic release of chemoattractive factors from the thymus. We further postulate that gate-opening reflects the expression of a unique pattern of chemokine/adhesion/ECM molecules by PCVs in the C-M junction of the thymus; and that gate-closing is initiated and maintained by TN1 and TN2 thymocytes, possibly by an IL-7 dependent mechanism.
Four specific aims are proposed to test these hypotheses: 1) to determine if the periodic release of prothymocytes from the BM is associated with their cyclical generation; 2) to determine if prothymocyte generation and mobilization is affected by diffusible chemotactic factors from the thymus; 3) to identify and characterize the intrathymic gates and niches for prothymocytes; 4) to determine which stages of early thymocytopoiesis are associated with gate opening and closing. The kinetics of prothymocyte proliferation in BM will be determined by BrdU-induced suicide and by DNA staining of purified prothymocytes with Hoechst 33342. The influence of the thymus on the kinetics of prothymocyte generation and mobilization will be studied in nu/nu and adult thymectomized, as well as normal, mice; as will the effects of thymus-derived chemoattractant factors and antibodies thereto. Purified prothymocytes from GFP transgenic mice will be used to identify binding sites (putative IMV-gates) along PCVs in the thymus of sublethally-irradiated mice, as well as the localization of these cells in putative IMN-niches. The gene expression profile of chemokine/adhesion/ECM molecules of purified high endothelial cells (HECs) from the prothymocyte-receptive LNs of Oncostatin-M and LIF transgenic mice will be determined by RNA hybridization. The involvement of candidate molecules in prothymocyte recruitment will be verified by antibody-staining/blocking studies, and the phenotype of these altered LN HEVs will be compared with that of the PCVs in the receptive thymus. The minimal cellular requirements for IMV-gate opening and closing, and the possible role of IL-7, wilt be determined by Lv. and Lt. adoptive transfer of prothymocytes from CD3 transgenic, RAG.-/-. and IL-7Ra -/- mice, and by the i.t. transfer of purified thymocyte subsets from WT mice. These results will provide basic information into the thymus-BM regulatory loop for prothymocytes, and may also have therapeutic implications regarding selective or enhanced T cell reconstitution, gene therapy, and classification/treatment of immunodeficiency disorders.
| Li, JiChu; Park, JooHung; Foss, Deborah et al. (2009) Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus. J Exp Med 206:607-22 |
| Goldschneider, Irving (2006) Cyclical mobilization and gated importation of thymocyte progenitors in the adult mouse: evidence for a thymus-bone marrow feedback loop. Immunol Rev 209:58-75 |
