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Different Subpopulations Of Developing Thymocytes Are Associated With Adherent (Macrophage) Or Nonadherent (Dendritic) Thymic Rosettes

Ken Shortman, David Vremec

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Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8+CD3++and CD4-CD8+CD3++mature thymocytes. M-ROS were enriched in CD4-CD8-thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROSassociated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.