SummaryA new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with antiimmunoglobulin-coupled magnetic beads. This enriched population (~80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCK)-CD3 complex, and having TCK/3 and 3" genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T ceU markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8ce mRNA. Thymic DC presented both the CD8 c~ and/3 chains on the cell surface (Ly-2+3+), although the ce chain was in excess; the splenic DC expressed only the CD8 ol chain (Ly-2 + 3-). h is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function. D endritic cells (DC), 1 first described by Steinman et al.(1, 2), are a minor population of irregularly shaped cells in lymphoid organs, distinguishable from both lymphocytes and macrophages. DC constitutively express high levels of both class I and class II MHC antigens (1, 2). They are highly efficient at presentation of antigen and stimulation of T lymphocytes (1-7). Those within the thymus are believed to effect deletion of developing T cells with self-reacting potential (8, 9). The surface antigenic pattern of thymic DC has been shown to differ somewhat from that of splenic DC (10-12), but it is not dear whether this implies the cells have a different origin, or a different function, or whether they simply represent different development states of the same functional lineage. There is evidence that it is the developmental state of the T cell that determines whether the T cell-DC interaction leads to T cell proliferation or to T cell death (13). As part of a study of the interaction between developing T cells in the thymus and thymic stromal elements (14), we have isolated thymic DC and compared them with those isolated from the spleen. The usual procedure for isolating DC involves enrichment in a low buoyant density fraction by centrifugation, followed by selection as cells that show an initial adherence but then release from the vessel surface when cultured overnight (10).We have used such a procedure to isolate DC from mouse thymus (15). However, we were con...

We have used intrathymic injection of fluorescein isothiocyanate to label thymocytes in situ. The method gives random labeling of the thymocyte population and so can be used to quantitate the extent of migration of cells from the thymus to the periphery. Migrant cells can be visualized in frozen sections or cell suspensions of peripheral organs by their fluorescence. Our data show that in young adults, about 1% of thymocytes leave the thymus per day. Since the bulk of thymocytes turn over every 5 to 7 days, this indicates that the vast majority (95%) of thymocytes die within the thymus. Cells that do leave the thymus, go mainly to the T areas of lymph nodes, spleen and Peyer's patches. Migrants are extremely rare in bone marrow, gut and liver. Migration is about the same in neonates as in adults relative to the size of the thymus, but is considerably lower in older animals where it is only about 0.1% of thymocytes per day at the age of six months.

We have reexamined the balance between cell birth, cell maturation, and cell death in the thymus by labeling dividing thymocytes and their progeny in vivo with [3H]-thymidine, isolating clearly defined subpopulations by fluorescence-activated cell sorting, and determining the distribution of label by autoradiography. When mature thymocytes were precisely defined (as CD4'CD8-CD3' or CD4-CD8' CD3+) and separated from immature single positives (CD4+CD8-CD3-and CD4-CD8+ CD3-), a lag was observed in the rate of entry of H3lIthymidine into mature cells. Thus, many of the mature thymocytes appear to derive from a small nondividing cortical thymocyte pool, rather than originating directly from the earliest dividing CD4+CD8+ blasts. There was little evidence for cell division during or after mature thymocyte formation, suggesting a one-for-one differentiation from cortical cells rather than selective clonal expansion. The rate of production of mature single positive thymocytes agreed closely with estimates of the rate of export of mature T cells from the thymus and was only 3% of the rate of production of doublepositive cortical thymocytes. This was compatible with a stringent selection process and extensive intrathymic cell death and suggested that no extensive negative selection occurred after the mature cells were formed.The majority of CD4+CD8+ (double positive) cortical thymocytes are believed to die in the thymus after a short life-span (1-5). However, some do mature into CD4+CD8-and CD4-CD8+ (single positive) medullary thymocytes through a process of positive selection by self major histocompatibility complex (MHC) antigens (6-15). The number entering the mature state is reduced by a process of negative selection, which eliminates cells with excessive direct reactivity with self-MHC and self-antigens (8,9,12,13,(16)(17)(18)(19). Cortical thymocytes that are not rescued by the positive selection process presumably also die intrathymically of neglect.Kinetic studies on T-lymphocyte development in the thymus originally presented the paradox of a very rapid rate of thymocyte birth (equivalent to one-third of all thymocytes per day) (1,4) compared to a slow rate of export to the periphery (equivalent to 1% of all thymocytes per day) (3, 4). The conclusion from the balance sheet was that most cells born in the thymus die in the thymus. A series of studies comparing the relative retention in the thymus of iododeoxyuridine and thymidine after their incorporation into thymocyte DNA had seemed to provide direct evidence for this intrathymic death (19-21); however, deiodination, rather than cell death and nucleoside reutilization, appears to have been the real explanation of these results (22). Since the balance sheet aspects remain the primary argument for extensive intrathymic death, it is important to recheck the evidence for thymocyte overproduction and to determine whether it is the mature or the immature subpopulations that are involved.The uptake of label into the DNA of cells in the S phase of the cell cyc...

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A continuous but low input of stem cells or 'prothymocytes' is necessary to maintain T-cell development in the adult thymus, but the colonizing cell has not been characterized. Precursors of T cells have been found in the minor CD4-8- population of thymocytes, but even the earliest cells of this population already have partially rearranged T-cell antigen receptor (TCR) genes. We now demonstrate that the thymus contains a minute population of lymphoid cells similar in some but not all respects to bone marrow-derived haemopoietic stem cells. This population has TCR genes in a germline state. It gives a slow but extensive reconstitution of both alpha beta and gamma delta lineages on transfer into an irradiated thymus, with kinetics indicating that it includes the earliest intrathymic precursor cells so far isolated. Surprisingly, these cells express low surface levels of the mature T-cell marker CD4.

Recent studies have opened the possibility that quiescent, G 0

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SummaryA new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from muhipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.