Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation.

Precursor CD4-CD8- (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta-selection subsequently expand and differentiate into immature CD4+CD8+ (DP) thymocytes. The cell subsets corresponding to the successive steps of this transition can be defined in terms of CD44 and CD25 expression. We partially synchronized the differentiation process by eliminating cycling cells with the anti-mitotic agent demecolcine. Using in vivo pulse labeling with bromodeoxyuridine, we determined the order of entry into DNA synthesis of the different DN and transitory (CD4-/lo CD8+) cell subsets. Two independent proliferation phases were identified. The first cells to enter the cell cycle were CD44-CD25lo, and CD4/CD8/TCR-/BrdU four-color staining showed that they all expressed a low density of the TCR-beta chain, an element of the pre-TCR (the TCR-alpha locus is still in germ-line configuration at this stage). Cycling of CD44+CD25+ cells was detected later, and no starting point was observed at the CD44-CD25hi stage. CD8 expression was immediately detectable in cycling cells, but they took 24 h to reach the DP stage. The study of TCR-Calpha-deficient mice showed that beta gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN-DP transition. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of pre-TCR expression.

Regulation and kinetics of premigrant thymocyte expansion.

Normal mature thymocytes proliferate before emigrating to the periphery, and continuous bromodeoxyuridine labeling showed that more than 30 % of fully mature thymic emigrants have replicated DNA in the 24 h before exit. The percentage of DNA-synthetizing single-positive (SP) thymocytes is transiently augmented during the postnatal period, with peaks on days 2 and 4 for CD4 and CD8 cells, respectively. Similar kinetics were observed in mouse chimeras made by transfer of normal bone marrow cells into RAG-2-deficient mice. These data show that proliferation of mature thymocytes is developmentally regulated. The proliferation peaks (on days 16 and 18 post transfer) observed in simple bone marrow chimeras were abolished when lymph node T cells were mixed with the bone marrow cell inoculum, suggesting that the peripheral pool controls the late thymic expansion. The phenotype of cycling SP thymocytes is atypical: they do not regulate activation and adhesion surface molecules like peripheral activated T cells.

Distribution of cycling T lymphocytes in blood and lymphoid organs during immune responses.

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vbeta8+ T cells showed a peak of DNA synthesis 16-24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vbeta8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vbeta6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-gamma inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.

Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency.

We recently characterized a CD4+ T cell population expressing the IL-2R alpha chain (CD25), producing IL-10 and resisting clonal deletion induced by viral superantigen (vSAG) encoded by mouse mammary tumor virus [MMTV(SW)]. We now report that these apoptosis-resistant cells are generated in the thymus but not from the immature CD4+ CD8+ thymocytes. They migrate from the thymus and are found in the periphery from at least the 10th day of life, after which they expand with the same kinetics in normal and MMTV(SW)-infected mice. Their strong capacity for expansion in the periphery makes this population insensitive to thymectomy in adulthood. CD4+ CD25+ cells were totally dependent on exogenous IL-2 for growth in vitro and in vivo, and were missing in IL-2 knockout (KO) mice. The absence of this population and/or an inability to produce IL-10 may be the missing link between IL-2R alpha KO, IL-2 KO and IL-10 KO mice, which all die of inflammatory bowel disease.

No evidence for proliferation in the blood CD4+ T-cell pool during HIV-1 infection and triple combination therapy.

OBJECTIVE: To evaluate the role of cell proliferation in peripheral blood lymphocyte (PBL) dynamics during HIV infection and potent antiretroviral therapy including protease inhibitors. DESIGN: Transverse study of 150 patients at different stages of infection. Longitudinal study of 50 patients on triple combination antiretroviral therapy with 9-month follow-up. METHODS: Ex vivo incubation of fresh PBL with the DNA biosynthetic marker bromodeoxyuridine (BrdU). Flow cytometric analysis of cell phenotypes and BrdU incorporation. Parallel determination of plasma virus load and CD4+ cell counts. RESULTS: Percentages of BrdU+ B and T lymphocytes found in patients with asymptomatic HIV infection were not different from the low values found in HIV-seronegative controls, and were not correlated with the CD4+ cell count. DNA synthesis increased significantly only during acute opportunistic infections occurring in patients with high plasma viral load and fewer than 100 x 10(6) CD4+ cells/l. Triple combination therapy induced a decrease of plasma virus load and a rise of CD4+ cell counts, whereas BrdU incorporation remained low or decreased. CONCLUSION: Proliferation of peripheral blood T cells observed at late stages of HIV infection corresponds to a response to opportunistic infections. Apart from these particular cases, proliferation in this compartment does not appear as a critical parameter of CD4+ cell kinetics during chronic HIV infection and potent therapy.

Expansion of mature thymocyte subsets before emigration to the periphery.

A small population of DNA-synthesizing mature thymocytes could be defined by analyzing cell surface markers and 5-bromo-2'-deoxyuridine (BrdUrd) labeling by four-color cytofluorometry. These cells have a completely mature phenotype (CD4- CD8+ or CD4+ CD8- TCR(high), HSA-, Qa-2(high)) and expand only weakly after BrdUrd incorporation. They recovered immediately in total number and in DNA synthesis rate after treatment with the antimitotic drug demecolcin, thus much faster than immature CD4+ CD8+ thymocytes. These data demonstrate the existence of a late intrathymic expansion phase, independent of that of developing CD4+ CD8+ immature cells, and involving phenotypically mature cells renewed each day. In mixed chimeras prepared by transfer of bone marrow and lymph node cells into RAG-2(-/-) mice, all cycling mature thymocytes were bone marrow derived. They are thus produced in situ and do not correspond to peripheral T cells reentering the thymus. Double FITC/BrdUrd detection showed that a high proportion (10-20%) of recent thymic emigrants were BrdUrd+ just postcycling cells and that around 50% of cycling mature thymocytes are just ready to emigrate to the periphery in the few hours after DNA synthesis. The late intrathymic expansion phase demonstrated here increases the daily thymic cell export by at least 30%. It could play a role in the adjustment of the T cell repertoire before emigration and in the regulation of the thymic cell output into the peripheral T cell pool.

T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity.

We found that T cells recognizing viral superantigen (vSAG) can be subdivided into two distinct functional subsets based on IL-2R alpha (CD25) expression. CD4+Vbeta6+CD25- and CD4+Vbeta6+CD25+ T cells were sensitive to vSAG activation. When obtained from BALB/c(SW) mice, both subsets were infected and capable to induce the tolerance process when transferred into noninfected recipients. However, in contrast to CD4+Vbeta6+CD25- cells, which were gradually deleted in MMTV(SW)-infected mice, the pool of CD4+Vbeta6+CD25+ lymphocytes was constant even at the end of the deletion process, and maintained a limited reactivity to vSAG-induced activation. The constant number of Vbeta6+CD25+ observed in infected mice could not be explained by their rapid turnover (deletion and renewal), as their proliferative rate measured by BrdU incorporation was similar in infected and naive mice, as well as in virus-nonspecific (Vbeta8.2+) cells. Neither was the Vbeta6+CD25+ subset dependent on vSAG activation since it was also present in MMTV-free mice and was not generated from Vbeta6+CD25- cells upon in vivo vSAG stimulation. Vbeta6+CD25+ T cells constitutively expressed IL-4 and IL-10 mRNA. IL-10 has been shown to be associated with viral, bacterial, and parasitic infections. This permanent CD25+ subpopulation may play a role in the control of viral infection and tolerance induction via vSAG recognition and IL-10 production.

In vivo T cell response to viral superantigen. Selective migration rather than proliferation.

Superantigens induce T cell activation and proliferation in vitro, and some also induce cell activation in vivo. MMTV(SW) is an infectious mouse mammary tumor virus (MMTV) encoding a superantigen with the same Vbeta specificity as MIs-1a (Mtv-7), which induces a strong local response in vivo. injection of MMTV(SW) into mouse footpads leads to accumulation of superantigen-reactive T cells (Vbeta6+CD4+) and B cells in the draining lymph nodes (LN). We investigated the kinetics of this cell accumulation by measuring cell activation (blastogenesis, CD25 and CD69 expression), cell migration (using syngenic FITC-labeled CD4+ cells and L-selectin detection), and cell proliferation (using in vivo labeling with bromodeoxyuridine). Specific T cells selectively migrated to the draining LN. Accumulating Vbeta6+CD4+ T cells were large CD69+ cells, but remained CD25 negative and showed down-regulated L-selectin expression. Their DNA synthesis rate, studied by pulse labeling and continuous administration of bromodeoxyuridine, was increased, but remained too low to explain the draining LN hyperplasia. These data show that the local T cell response to MMTV(SW) mainly consists of selective migration followed by local activation of reactive T cells, and that cell proliferation is only a minor component of the response. By contrast, the optimal dose of staphylococcal enterotoxin B that, nevertheless, leads to a lower reactive T cell accumulation in the draining LN induces a very high proliferation rate.

CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway.

Thymocytes with a low expression of CD4 and an intermediate density of the TCR (CD4low TCRint) were analyzed for phenotype, MHC dependence, production kinetics, and TCR repertoire to investigate their position in the intrathymic T cell maturation process. Comparison of normal and MHC-deficient mice showed that the CD4low TCRint cell subset was MHC class II dependent, as this subpopulation could not be defined in MHC class II- or double (class I and II)-deficient mice. These thymocytes were heat-stable Aghigh and CD69+, thus immature and recently engaged in a TCR interaction, probably with MHC class II molecules. Their generation kinetics were studied in two systems: development of exogenous bone marrow cells transferred into RAG-2-/- mice, and pulse labeling with bromodeoxyuridine. In both systems, CD4low TCRint cells were produced well before CD4low TCRhigh cells, the direct precursors of CD8 single-positive cells. Their production paralleled that of CD4high TCRint cells, but they were different than these thymocytes in their smaller cell size. Moreover, they had the same V beta 6 frequency in Mls-1a and Mls-1b mice, suggesting that these cells could be undergoing a negative selection process. The data here clearly demonstrate that CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway, and suggest that these cells could represent a MHC class II-restricted dead-end subset.

Thymic medulla epithelial cells acquire specific markers by post-mitotic maturation.

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2/-mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4+ single-positive cells were generated, from resting CD4+CD8+ cells after day 15. During this day 10-15 period, many epithelial cells positive for cortex-specific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day 15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd+. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.

Stochastic coreceptor shut-off is restricted to the CD4 lineage maturation pathway.

Kinetics of mature T cell generation in the thymus of normal or major histocompatibility complex (MHC) class I- or II-deficient mice were studied by the bromodeoxyuridine pulse labeling method. As previously described, the early activation and final maturation phases were found to be synchronous for the two T cell lineages, but CD4+8- cells were generated faster than CD4-8+ cells in MHC class I- and II-deficient mice, respectively. CD8 downregulation started on day 2 after cell proliferation even in the absence of MHC class II expression. CD8 downregulation thus appears to be stochastic at its beginning. By contrast, CD4 shut-off was found totally instructive, as the generation of CD4lo8+ cells with a high TCR density was not observed in class I-deficient mice. The analysis of the V beta 14 TCR frequencies in CD4/8 subsets in normal and MHC-deficient mice confirmed that CD4 and CD8 generation pathways are not symmetrical. These findings show that commitment towards the CD4+8- or CD4-8+ phenotype is controlled at the CD8lo step for the former and at the CD4+8+ double-positive stage for the latter.

Cell expansion and growth arrest phases during the transition from precursor (CD4-8-) to immature (CD4+8+) thymocytes in normal and genetically modified mice.

T cell early precursors belong to the CD3-CD4-CD8- triple negative (TN) thymocyte population that can be subdivided on the basis of CD44, CD25, and heat-stable Ag (HSA) expression. The kinetics and precursor product relationships of these subsets, as well as of the CD4/8low intermediates, were studied by using pulse labeling with bromodeoxyuridine (BrdUrd). The highest frequencies of DNA-synthesizing cells were found in CD44+CD25+ and CD44-CD25low or CD25- subsets. The major TN cell type (CD44-CD25high), as well as CD44+ CD25-HSAlow early precursors, contained a majority of resting cells. RAG-2-/- mice contained less cells in DNA synthesis than normal mice, and CD44-CD25-/low cells were absent. In female mice transgenic for the anti-HYTCR, CD44-CD25high cells were almost all cycling, but a high percentage of resting cells was found in CD44-CD25- cells. In days following the BrdUrd pulse, there was a reduction in the number of BrdUrd+ cells in most subsets, with the exception of the labeled CD44-CD25high cells that showed a bell-shaped curve. The kinetics and cell size evolution suggest that the majority of these cells do not give rise to CD4+CD8+ cells. In RAG-2-/- cells, the block at the CD44-CD25high stage involved all cells. In TCR transgenic (Tg) mice, no block was seen at the CD44-CD25high stage, suggesting that early expression of a complete TCR receptor precludes the normal selection step. However, another block in the differentiation process was observed at the CD44-CD25- step in TCR Tg mice, suggesting an additional selection point.

Endogenous granulocyte-macrophage colony-stimulating factor is involved in IL-1- and IL-7-induced murine thymocyte proliferation.

We have reported previously that IL-1 induces murine thymocyte proliferation in the absence of artificial comitogens, provided that the cells are cultured at high densities. In the present study, we show that, in these conditions, TdR uptake in response to IL-1 is diminished significantly by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) Abs. Indeed, a substantial production of this growth factor occurs when thymocytes are cultured in the presence of IL-1. Maximal GM-CSF levels are attained within 3 days of culture, and mRNA expression is detected after a 48-h stimulation. Both GM-CSF production and IL-1-induced thymocyte proliferation are decreased considerably by the depletion of I-A+ Mac-1+ accessory cells. Yet, addition of exogenous GM-CSF to accessory cell-depleted thymocytes does not restore the proliferative response to IL-1 alone, suggesting the implication of another accessory cell-derived mediator. Our data design IL-7 as the endogenous factor required in our culture system because: 1) GM-CSF can reverse the decrease in the proliferation after accessory cell depletion when IL-7 is provided together with IL-1, and 2) the proliferative response to IL-1 plus IL-7 is diminished as much by neutralization of GM-CSF by its specific Abs as by accessory cell removal (approximately 30%). Finally, the cells responding to IL-1 + IL-7 were identified as mature CD4-CD8-TCR+ thymocytes by the use of bromodeoxyuridine (BrdUrd), suggesting that the GM-CSF produced by thymic accessory cells in response to IL-1 participates in IL-7-dependent, intrathymic expansion of the CD4-CD8-TCR+ compartment.

Interleukin 7 induces preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4+8- murine thymocytes positively selected by class I molecules.

We analyzed the phenotype and V beta-T cell receptor (TCR) repertoire, together with interleukin 7 receptor (IL-7R) expression in unfractionated thymocytes stimulated in vitro with IL-7. This culture system results in a specific proliferation of mature thymocytes belonging to the CD3+CD4-, CD4+8-, and CD4-8+ subsets. IL-7 induced a preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4-8- thymocytes. This phenomenon is not observed in beta 2-microglobulin-deficient mice, showing that a fraction of CD4+8- thymocytes, enriched in V beta 8.2+ cells, is selected by class I molecules in normal mice, as are a large proportion of CD4-8- alpha beta TCR+ thymocytes. Our findings also establish that IL-7 plays a major role in the expansion of rare thymocyte subsets, which could exert important functions in inflammatory and immune responses.

Production, selection, and maturation of thymocytes with high surface density of TCR.

The main steps in intrathymic T cell differentiation have been defined using bromodeoxyuridine as a postmitotic cell tracer. Thymocytes with a high surface expression of the TCR are generated in the first 24 h after DNA synthesis. The phenotype of these TCR(high) cells was studied during 10 days by using pairs of surface markers associated with BrdUrd. During the first 2 days, TCR(high) cells were of the CD4+CD8+HSA(high) phenotype, transiently expressed the early activation marker CD69, and contained a high percentage of cycling cells. This activation step preceded the transition from CD4+CD8+ to CD4+CD8- and then to CD4-CD8+ cells, followed by progressive HSA down regulation and increase in the expression of H-2K, Qa-2, and CD45RB. The phenotypic maturation was completed in 9 days. In Mls-1a mice, negative selection of V beta 6+ cells was observed at the earliest step of TCR(high) cell generation, and positive selection of V beta 8.2+ and V beta 14+ cells took place later and was correlated to the activation step. These data suggest that high TCR expression and cell activation are necessary for positive selection and subsequent T cell maturation.

Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'-deoxyuridine-pulse-labeled thymocytes. Correlation with T cell receptor expression.

"In vivo" kinetics of T cell differentiation and TCR expression in the normal murine thymus were re-evaluated using a new technique for simultaneous detection of bromodeoxyuridine and two surface markers. The transition from CD4-8- precursors to CD4+8+ immature cells was directly observed during cell proliferation, and shown to proceed through transitory intermediates expressing no or low amounts of CD4. CD3-TCR expression also started during this transition and resulted in the production of a majority of TCRlo cells but also of a significant number (1 to 2 x 10(6) of TCRhi immature (heat-stable Ag+) thymocytes. After cessation of proliferation, the maturational transition from CD4+8+ to CD4+8- and CD4-8+ (in this order) was restricted to TCRhi cells produced during CD4+8+ cell generation. The acquisition of the single positive phenotype preceded HSA down-regulation, suggesting that maturation of TCRhi thymocytes proceeds in two separate steps. The major TCRloCD4+8+ subset appeared a dead end subset and showed no up-regulation of TCR expression at any time.

Phenotype analysis of cycling and postcycling thymocytes: evaluation of detection methods for BrdUrd and surface proteins.

We present a comparison of two different methods for simultaneous detection of bromodeoxyuridine and cell surface markers. Both methods use enzymatic generation of single-strand DNA with nuclease. The biological system used is the murine thymus, in which in vivo DNA synthetizing cells were labeled by injection of BrdUrd and analyzed at different time points after the nucleoside pulse. The surface proteins detected were CD4 and CD8 differentiation markers and the T-cell receptor. Extraction of DNA-associated proteins with 0.1N HCl and detergent is necessary for the action of EcoR1 and Exonuclease III, but this treatment destroys phycocyanins and induces cell aggregation, as shown using the doublet-discrimination module. For DNAse I action, cells could be treated with paraformaldehyde and a low concentration of Tween 20, and this treatment was adequate for surface staining preservation (even with phycocyanins) and BrdUrd detection. Both methods were adequate for cell cycle studies, but only 7-amino-actinomycin D could be used as total DNA dye after DNAse action, and good results needed long (48-72 h) incubation in the fixative-detergent mixture. The DNAse I method now allows three-color staining (two surface markers and Brd-Urd), analyzed in a one laser-cytometer for the study of the phenotype of cycling cells, and of their progeny, in vivo and in cell cultures. It also allows the quantitative analysis of cell surface receptor densities in conditions similar to fresh cells.

A novel CD45RA+CD4+ transient thymic subpopulation in MRL-lpr/lpr mice: its relation to non-proliferating CD4-CD8-CD45RA+ tumor cells.

MRL-lpr/lpr mice have hypertrophied lymph nodes comprising CD4-CD8- T cells. In addition, they contain CD4+CD8- T cells co-expressing the CD45RA marker. The correlation between these two subpopulations has been difficult to assess. We analyzed the expression of CD45RA (with the RA3-2C2 antibody) in various thymic and peripheral T cell subsets, using three-color immunofluorescence. We showed that in lpr mice (i) a transient CD4+CD8- thymic subset co-expresses CD45RA during the course of the disease, and (ii) thymic as well as peripheral CD4-CD8- and CD4+CD8- T cells brightly express CD45RA; furthermore (iii) in the lymph nodes, during lymphadenopathy, CD4+CD8-CD45RA+ T cells show a broad range of the CD4 fluorescence intensity, and (iv) the increase in MHC class II expression is restricted to CD45RA-T cells of the thymus and lymph nodes of lpr mice. Taken together, these data suggest that the CD4+CD8-CD45RA+ population might generate the CD4-CD8- tumor cells. In addition, using the bromodeoxyuridine labeling technique, we demonstrate that these cells are not the result of increased proliferation.

Thy-1 modulation and cell proliferation at early steps of intrathymic bone marrow cell differentiation.

Intrathymic (IT) transfer of bone marrow (BM) precursor cells in sublethally irradiated hosts has been widely used to study T cell differentiation and maturation. In this report we have used double congenic mice Ly 5.1 Thy 1.1 (host) and Ly 5.2 Thy 1.2 (donor) and detected cycling Ly 5.2+ BM cells by in vivo bromodeoxyuridine incorporation, before induction of the Thy 1.2 antigen. Until Day 9 post-transfer, some donor type cells express a high level of Thy 1.2 together with macrophage and granulocyte markers. A few days later, a Thy 1.2low population transiently B220+ was detected. Thereafter, donor type cells expressed an intermediate Thy 1.2 brightness; this population then persisted and surpassed the other subsets. Our findings permitted to establish a relationship between cell cycle and Thy 1 fluorescence intensity according to the sequence: Thy 1low resting, Thy 1low cycling, Thy 1high cycling, Thy 1high resting. Moreover, we have shown that cells from the myeloïd and B lineages can, in vivo, transiently express the Thy 1 antigen, develop and differentiate within the thymus microenvironment.

Accumulation of bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: minimal estimates of production and turnover rates of mature lymphocytes.

Daily lymphocyte production in both central and peripheral lymphoid organs was evaluated by associating in vivo incorporation of bromodeoxyuridine (BrdUrd) with cell surface labeling and multi-parameter flow analysis. At least 10% of mature T and B lymphocytes are generated every 24 h. The kinetic behavior of these cell populations differs, however, in that mature B cells are generated predominantly in the precursor compartments of the bone marrow, while most mature T cell generation occurs at the periphery. Therefore, peripheral expansion is the major mechanism of mature T cell production in the adult mouse. By following the accumulation of BrdUrd-labeled cells in peripheral lymphoid organs we found that the progeny of the daily lymphocyte production was sufficient to renew 30%-40% of all peripheral T and B cells every 48 h, demonstrating a high turnover rate of mature lymphocytes. We also examined the conditions of BrdUrd labeling of cycling cells in vivo. We found that while greater than 90% of bone marrow and thymus cells in S phase were labeled with a single injection of BrdUrd, in peripheral lymphoid compartments 70% of T and B cells in S failed to incorporate BrdUrd. Particular schedules of BrdUrd administration were required to overcome the low labeling efficiency of mature cells in vivo. Prolonged BrdUrd administration, however, had toxic effects on resident cells. The low labeling efficiency of BrdUrd incorporation by mature cells, as well as its potential toxicity during prolonged administration, may explain controversial results obtained by the different strategies used to study lymphocyte population dynamics.