Kinetics of mature T-cell development in the thymus.

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 [3H]thymidine 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 double-positive 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.     

Interactions between double positive thymocytes and high affinity ligands presented by cortical epithelial cells generate double negative thymocytes with T cell regulatory activity

Previous studies on thymocyte differentiation by using reaggregate cultures (RC) of double positive T cell receptor (TCR) transgenic thymocytes and the thymic epithelial cell line ANV indicated that low concentrations of high affinity ligands for the TCR were efficient inducers of thymocyte maturation to CD4 single positive (SP) functional cells. In this study, it is demonstrated that, when high concentrations of high affinity ligands are used in this RC system, double positive (DP) cells down-modulate expression of both coreceptors and that, as a result, large numbers of double negative (DN) cells are generated. These DN cells proliferated modestly in response to stimulation by antigen, and this response was considerably augmented by the addition of IL-2 to the cultures. Notably, these antigen-stimulated DN cells produced large amounts of IL-10. When the DN cells generated in RC were cocultured with naive TCR transgenic T cells in the presence of antigen, they suppressed the proliferative response of the naive T cells. Thus, high affinity ligands, when presented to DP thymocytes by cortical thymic epithelial cells in reaggregate cultures, rather than causing deletion of the immature thymocytes, induce their differentiation into immunoregulatory DN cells, suggesting a distinct mechanism by which self tolerance may be maintained.     

Notch ligands Delta 1 and Jagged1 transmit distinct signals to T-cell precursors

Signaling through the Notch pathway plays an essential role in inducing T-lineage commitment and promoting the maturation of immature thymocytes. Using an in vitro culture system, we show that 2 different classes of Notch ligands, Jagged1 or Delta1, transmit distinct signals to T-cell progenitors. OP9 stromal cells expressing either Jagged1 or Delta1 inhibit the differentiation of DN1 thymocytes into the B-cell lineage, but only the Delta1-expressing stromal cells promote the proliferation and maturation of T-cell progenitors through the early double-negative (DN) stages of thymocyte development. Whereas the majority of bone marrow-derived stem cells do not respond to Jagged1 signals, T-cell progenitors respond to Jagged1 signals during a brief window of their development between the DN1 and DN3 stages of thymic development. During these stages, Jagged1 signals can influence the differentiation of immature thymocytes along the natural killer (NK) and gamma delta T-cell lineages.     

Data base analysis of protein expression patterns during T-cell ontogeny and activation.

We have developed a data base of lymphoid proteins detectable by two-dimensional polyacrylamide gel electrophoresis. The data base contains two-dimensional patterns and derived information pertaining to polypeptide constituents of unstimulated and stimulated mature T cells and immature thymocytes, single-cell-derived T- and B-cell clones, leukemia cells, and lymphoid cell lines. Using this data base, we have compared the protein constituents of mature T cells and immature thymocytes before and after mitotic stimulation. A subset of polypeptides that are induced in mature T cells following mitotic stimulation were found to be constitutively expressed in immature thymocytes. Other polypeptides exhibited differences in their expression between mature and immature thymocytes in a manner unrelated to proliferation. The identity of several constitutively expressed or mitotically induced proteins in lymphoid cells was established by microsequencing. These initial findings point to significant differences in the molecular pathways leading to proliferation between mature and immature T cells. The construction of this database should facilitate further studies of lymphoid differentiation and function.     

Combined and sequential effects of human IL-3 and GM-CSF on the proliferation of CD34+ hematopoietic cells from cord blood

The proliferative effects of recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were investigated in semi-solid and liquid cultures of purified CD34+ hematopoietic cells obtained from umbilical cord blood. No important differences in overall cloning efficiencies in response to IL-3 or GM- CSF were observed in semi-solid medium in the presence of erythropoietin (Ep). However, GM-CSF was less effective for the development of erythroid bursts (BFU-E), and only IL-3 was observed to induce significant numbers of mixed-erythroid colonies (E-MIX). Both IL- 3 and GM-CSF also induced proliferation of CD34+ in liquid cultures. Proliferative responses to IL-3 were found to be more rapid and stronger than to GM-CSF, although the number of initial responsive cells as judged by autoradiography were comparable. Enhanced proliferation of CD34+ cells both in semi-solid and liquid cultures was obtained in the presence of combinations of IL-3 and GM-CSF. The responses observed were less than additive, with the exception of the development of eosinophil colonies and clusters, where IL-3 and GM-CSF were found to act synergistically. In secondary cultures, proliferative responses to GM-CSF were strongly enhanced by preculture of CD34+ cells in IL-3 for four to 11 days, and to a lesser extent by preculture in GM- CSF. Finally, responses to IL-3 were not affected by preculture of CD34+ cells in the presence of GM-CSF. Our results indicate that there is a wide overlap of cells capable of proliferating either in response to IL-3 or to GM-CSF within the cord blood CD34+ compartment. However, differences in primary proliferation kinetics and increased responsiveness to GM-CSF following preculture suggest the importance of a sequential action of IL-3 and GM-CSF in the expansion of CD34+ cells.     

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.     

Differential expression of integrins on human thymocyte subpopulations

Integrins represent a candidate group of cell surface receptors that may control the homing and population of the thymus by T-cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. We have used multiparameter flow cytometric methods to characterize the expression of several members of the integrin family (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha L beta 2) on thymocyte subpopulations and have correlated integrin expression with other well-defined thymocyte differentiation markers. alpha 4 beta 1 was expressed by all thymocytes, but expression was highest on CD4-CD8- double-negative (DN) cells, high on CD+CD8+ double-positive (DP) cells, and lowest on mature single-positive (SP) cells, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were present on 13%, 63%, and 26% of thymocytes, respectively, with maximal levels of expression on DN and SP cells, and low levels of expression on DP cells. Simultaneous analysis of alpha 4 beta 1, alpha 5 beta 1, and CD3 expression suggested a pathway of T-cell differentiation in the thymus in which the majority of the DN cells were alpha 4 beta 1hi alpha 5 beta 1hi, the DP cells alpha 4 beta 1hi alpha 5 beta 1lo/-, and the most mature SP cells were alpha 4 beta 1int. The stage-specific expression of integrins strongly implies their functional involvement during T-cell maturation in the thymus.     

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.     

Immature T-cell clustering and efficient differentiation require the polarity protein Scribble

T-cell polarization is required for cell migration and cell-cell interactions, cellular behaviors crucial for lymphocyte differentiation. Despite expression of the epithelial polarity network in T cells, neither its contribution to thymocyte polarity nor its requirement during development is known. We report here that depletion of the polarity protein Scribble in hematopoietic progenitor cells results in inefficient T-cell development characterized by a partial developmental block during the early double-negative (DN) stage of differentiation. Scribble-depleted hematopoietic progenitor cells exhibit a delayed transition into late CD44(lo/-)CD25(+) DN3 cells, evidenced by the accumulation of early CD44(int)CD25(+) DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor β-positive DN3 cells are observed among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not exhibit compromised spontaneous motility, T-cell clustering and prolonged homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during T-cell migration or on the initiation of T-cell-T-cell interactions. Scribble is therefore a critical contributor to the clustering of immature T cells, an event shown here to be necessary for efficient developmental progression.     

A newly established murine immature dendritic cell line can be differentiated into a mature state, but exerts tolerogenic function upon maturation in the presence of glucocorticoid

The phenotype and function of murine dendritic cells (DCs) are primarily studied using bone-marrow-derived DCs (BM-DCs), but may be hampered by the heterogeneous phenotype of BM-DCs due to their differential state of maturation. Here we characterize a newly established murine DC line (SP37A3) of myeloid origin. During maintainance in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, SP37A3 cells resemble immature DCs characterized by low expression of major histocompatibility complex (MHC) II and costimulatory molecules and low T-cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype and activate naive T cells as potently as BM-DCs. Similar to BM-DCs, SP37A3 cells activated in the presence of dexamethasone-induced regulatory T cells, which were anergic upon restimulation and suppressed proliferation of naive T cells. This tolerogenic state was reflected by lower expression levels of costimulatory molecules and proinflammatory cytokines compared with mature cells, as well as up-regulated expression of FcgammaRIIB and interleukin-1RA (IL-1RA). SP37A3 cells were responsive to dexamethasone even when applied at later time points during activation, suggesting functional plasticity. Thus, DC line SP37A3 represents a suitable model to study functions of immature and mature as well as tolerogenic myeloid DCs, circumventing restrictions associated with the use of primary DCs and BM-DCs.     

Stage-dependent reactivity of thymocytes to self-peptide--MHC complexes

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature na?ve T cells in the periphery.     

Interleukin-7 receptor controls development and maturation of late stages of thymocyte subpopulations

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα–floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αβ T cells suppresses development of other cell lineages in thymus.     

Granulocyte/macrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a specific humoral growth factor that stimulates both neutrophilic granulocyte and macrophage production by bone marrow hematopoietic progenitor cells. GM- CSF also stimulates the proliferation and clonal growth of both tissue macrophages and blood monocytes. Although at low concentrations GM-CSF was unable to support the long-term growth of tissue macrophages, it greatly enhanced their responsiveness to macrophage CSF (M-CSF, or CSF- 1). This effect was also observed by treating macrophages with GM-CSF for a short time. GM-CSF did not compete with M-CSF for binding to M- CSF receptors nor was it inactivated by treatment with anti-M-CSF antiserum. Treatment of tissue macrophages with GM-CSF led to a rapid but transient downregulation of M-CSF receptors; prolonged incubation at 37 degrees C, however, resulted in a restoration and upregulation of M-CSF receptors. Identical effects were observed with both native or recombinant GM-CSF. This study suggests that GM-CSF regulates tissue macrophage production by two modes of action: (a) direct stimulation of macrophage proliferation, and (b) enhancement of their responsiveness to M-CSF.     

Age-associated changes in CD90 expression on thymocytes and in TCR-dependent stages of thymocyte maturation in male rats

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRalphabeta and (ii) Thy-1, which is supposed to be a regulator of TCRalphabeta signalling, and thereby the thymocyte selection thresholds. Since an essential role for TCRalphabeta signalling in the development of CD4+25+T(reg)-cells was suggested, the frequency of these cells was also quantified. We demonstrated that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive. Furthermore, we revealed that TCRalphabeta-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression of Thy-1 on TCRalphabeta(low) thymocytes entering selection processes. The diminished frequency of the post-selection CD4+8+ double positive (DP) cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational transition from the DP TCRalphabeta(high) to the SP TCRalphabeta(high) developmental stage, which is followed by an increase in pre-migration proliferation of the mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4+25+T(reg)-cells in aged rats, thus providing a possible explanation for the increased frequency of autoimmune diseases in ageing.     

Stromal cell-derived factor 1/CXCR4 signaling is critical for early human T-cell development.

The present study investigated the potential role of stromal cell-derived factor 1 (SDF-1) in human intrathymic T-cell differentiation. Results show that SDF-1 is produced by human thymic epithelial cells from the subcapsular and medullary areas, and its receptor, CXCR4, is up-regulated on CD34(+) precursor cells committed to the T-cell lineage. Chimeric human-mouse fetal thymus organ culture (FTOC) seeded with purified CD34(+) thymic progenitors and treated with neutralizing antibodies against SDF-1 or CXCR4 showed a significant reduction of the number of human thymocytes and an arrested thymocyte differentiation in the transition between CD34(+) precursor cells and CD4(+) immature thymocytes. SDF-1-treated FTOC showed an increase of human thymocyte numbers, mainly affecting the most immature subpopulations. Moreover, these results suggest that CXCR4/SDF-1 signaling is not critical for the CD34(+) cell precursor recruitment to the thymus. On the other hand, SDF-1 significantly increased the viability of CD34(+) T-cell precursors modulating the expression of BCL-2 and BAX genes, and stimulated the proliferation of CD34(+) thymic precursor cells, particularly in synergy with interleukin 7 (IL-7), but not with other cytokines, such as stem cell factor or flt3-ligand. Accordingly, only IL-7 was able to up-regulate CXCR4 expression on CD34(+) thymic progenitors. In addition, deprivation of SDF-1 partially inhibited human thymocyte expansion induced by IL-7 in human-mouse FTOC. This study indicates that SDF-1/CXCR4 signaling is required for the survival, expansion, and subsequent differentiation of human early thymocytes and identifies a new mechanism by which IL-7 mediates its effects on human thymopoiesis.     

Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.     

Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors

A microculture assay for murine granulocyte-macrophage colony- stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five- fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM- CSF for several months. Microassay results for several sources of GM- CSF at different stages of purification are compared to the results obtained from the soft agar assay.     

K channels are expressed early in human T-cell development.

Mature human T lymphocytes proliferate in response to the mitogen phytohemagglutinin (PHA), but immature thymocytes lacking the T3 receptor (T3- thymocytes) do not. Because functioning K channels are required for proliferation of mature T cells, we asked whether immunoincompetent T3- thymocytes lack normal K channels. We report that T3- thymocytes have a K+ current similar to that of mature peripheral T cells--that is, similar voltage dependence, activation and inactivation kinetics, and pharmacology. Moreover, the maximal specific K+ conductance is the same for both cell types, implying a similar density of activable channels in each cell. In assessing the functional responses of the channels to PHA, we found that the K+ current of immature and mature cells responds similarly to the mitogen. Responses near the threshold voltage for activating the K+ current were variable; the K+ conductance and rate of activation were increased, decreased, or unchanged after PHA treatment. For several cells, the voltage dependence of the conductance and activation kinetics was shifted in opposite directions. At more positive voltages, PHA consistently caused a 10-20% suppression of conductance that was not due to the addition of an inward current, to changes in the time course of activation or inactivation, or to changes in the steady-state level of inactivation. The effects of PHA on the K+ current cannot be explained by a simple shift in surface potential, as has been hypothesized to be involved in its triggering of T-cell proliferation. Taken together, our findings show K channels are expressed very early in T-cell differentiation, possibly before thymic processing, differential responses of the K+ current to PHA do not account for the failure of T3- thymocytes to proliferate, and changes in surface potential are probably not a necessary early event in activation of T cells by PHA.     

Homeostatic control of T-cell generation in neonates

T cells are produced through 2 mechanisms, thymopoiesis and proliferative expansion of postthymic T cells. Thymic output generates diversity of the pool, and proliferation achieves optimal clonal size of each individual T cell. To determine the contribution of these 2 mechanisms to the formation of the initial T-cell repertoire, we examined neonates of 30 to 40 weeks' gestation. Peripheral T cells were in a state of high proliferative turnover. In premature infants, 10% of T cells were dividing; the proliferation rates then declined but were still elevated in mature newborns. Throughout the third trimester, concentrations of T-cell-receptor excision circles (TRECs) were 10 per 100 T cells. Stability of TREC frequencies throughout the period of repertoire generation suggested strict regulation of clonal size to approximately 10 to 20 cells. Neonatal naive CD4+ and CD8+ T cells were explicitly responsive to IL-7; growth-promoting properties of IL-15 were selective for newborn CD8+ T cells. Neonatal T cells expressed telomerase and, in spite of the high turnover, built up a telomeric reserve. Thus, proliferative expansion, facilitated by increased cytokine responsiveness, and thymopoiesis complement each other as mechanisms of T-cell production in neonates. Maintaining optimal clonal size instead of filling the space in a lymphopenic host appears to regulate homeostatic T-cell proliferation during fetal development.