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The Journal of Immunology, 2001, 166: 5898-5908.
Copyright © 2001 by The American Association of Immunologists

Distinct Mechanisms Contribute to Generate and Change the CD4:CD8 Cell Ratio During Thymus Development: A Role for the Notch Ligand, Jagged1

Eva Jiménez*, Angeles Vicente{dagger}, Rosa Sacedón*, Juan J. Muñoz*, Gerry Weinmaster{ddagger}, Agustín G. Zapata* and Alberto Varas*

Department of Cell Biology, Faculties of * Biology and {dagger} Medicine, Complutense University, Madrid, Spain; and {ddagger} Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4+CD8- and CD4-CD8+ thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most conventional T lymphocytes fall into two main subsets, MHC class II-restricted CD4+ helper cells and MHC class I-restricted CD8+ cytotoxic cells. In normal adults, peripheral CD4+ T cells are more abundant than CD8+ T cells, and the relative proportions of these two lymphocyte subsets are finely controlled (1, 2, 3). Intraspecies differences in the size of the peripheral CD4 and CD8 T cell pools are genetically determined in humans and mice (4, 5). These distinct CD4:CD8 cell ratios observed in peripheral T lymphocytes have been reported to originate in the thymus (6, 7, 8). Nevertheless, the intrathymic mechanisms involved in determining the peripheral CD4:CD8 cell ratios seem to be species-specific. In adult rats, differences in CD4:CD8 cell ratio are MHC haplotype-dependent (8). On the contrary, allelic differences in the TCR{alpha} loci (6) and genetic variations in the process of CD4 vs CD8 lineage commitment (7) have been described to explain the distinct CD4:CD8 cell ratios in mice.

In contrast to adult animals, peripheral CD8+ T cells predominate over CD4+ T cells in young animals (9), but the origin of this difference has not been elucidated. Adkins (10, 11) correlated the occurrence of different CD4:CD8 cell ratios in fetal/neonatal and adult mice with distinct maturation potentials of both the embryonic and adult CD4-CD8- thymic precursors, and developmental differences in the thymic stromal environment. Likewise, Shortman and Wu (12) have postulated that the differential production of CD4+ and CD8+ T cells could reflect the presence or absence of TdT in the adult or fetal precursors, respectively, because the higher N region TCR diversity associated with the adult TdT+ precursors could lead to more MHC class II-restricted receptors appropriate for CD4+ T cells. It is also possible to postulate that some of the signals involved in CD4/CD8 lineage commitment (13, 14) could change throughout thymus development. The Notch family of receptors, which regulate cell fate decisions during the development of many cell lineages in both vertebrates and invertebrates (15, 16, 17), have been implicated in CD4 vs CD8 T cell lineage determination (18, 19). Remarkably, analyses of Notch1 expression during thymus development have shown that the highest levels are found in the fetal thymus, whereas Notch1 expression decreases in the neonatal/adult thymus (20, 21).

In this report, we demonstrate that, as in adult life, the fetal CD4:CD8 cell ratio is established in the thymus, and that the generation of distinct proportions of CD4+CD8- and CD4-CD8+ thymocytes in the fetal/neonatal and adult thymus is developmentally regulated by the thymic stromal environment. The Notch ligand Jagged1 is expressed by the thymic stroma suggesting that Notch signaling might regulate CD4:CD8 cell ratio. Using a reaggregation assay we show that interactions between Jagged-expressing cells, uncommitted double positive (DP)3 thymocytes, and thymic stromal cells alter the ratio CD4:CD8 consistent with a role for Notch signaling in this process. Our results also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth, mainly promoted by differences in the proliferative and emigration rates of the mature thymocyte subsets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Wistar Hannover rats were maintained in our animal facilities. Rat fetuses at days 19–21 of gestation were obtained from timed pregnancies. The day of finding a vaginal plug was designated day 0 of gestation.

Cytofluorometric analysis and cell sorting

Mouse anti-rat mAbs of the following specificities were used in our study: CD4 (OX35-FITC, -PE, or -CyChrome), CD8{alpha} (OX8-FITC, -PE, or PerCP), CD8{beta} (341-FITC or -biotin), and TCR{alpha}{beta} (R.73-FITC or -PE) were obtained from PharMingen (San Diego, CA). Two- and three-color flow cytometric analyses of lymphocytes were performed as described previously (22). For the phenotypic analysis of thymic epithelial cells, purified mAbs against rat MHC class II I-A like (OX6) and I-E like (OX17), and MHC class I molecules (OX18) were obtained from PharMingen. These unconjugated Abs were revealed with PE-conjugated F(ab')2 of rabbit anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Isotype-matched irrelevant Abs were used as negative controls to define background fluorescence. Cells were then treated with a FACS-permeabilizating solution (BD Biosciences, San Jose, CA), according to the instructions of the manufacturer, and stained with FITC-conjugated anti-cytokeratin (SigmaEspaña, Madrid, Spain). Stained cells were analyzed in FACScan andFACSCalibur flow cytometers (BD Biosciences) from the Servicio Común de Investigación, Faculty of Biology, Complutense University of Madrid. The data were analyzed using PC lysis and CellQuest research software (BD Biosciences).

Fetal and adult CD4+CD8+ (for the coreceptor re-expression assay) and CD4+CD8- and CD4-CD8+ thymocytes (for the cell cycle analysis) were electronically sorted from a thymocyte population stained with anti-CD4 and anti-CD8 mAbs. Purity of the sorted populations was 97–99%. Cell sorting was performed on a FACStarPlus (BD Biosciences) at the Centro de Citometría de Flujo, Complutense University of Madrid.

Cell cycle analysis

To determine the proportion of proliferating mature thymocytes, electronically sorted CD4+CD8- or CD4-CD8+ cells were stained with anti-TCR{alpha}{beta} for 30 min at 4°C. Peripheral lymphocytes were stained with either anti-CD4 or anti-CD8, and anti-TCR{alpha}{beta}. Thymocytes from reaggregation cultures were stained with anti-CD4 and anti-CD8. Cells were then fixed with 30% ethanol and incubated with 7-amino actinomycin D (7-AAD) as previously described (23). Analysis was conducted in a FACScan, using Cell Fit and PC lysis software.

Apoptosis assay

After cells were stained with either anti-CD4/anti-CD8, anti-CD8/anti-TCR{alpha}{beta}, or anti-CD4/anti-TCR{alpha}{beta} and washed twice with PBS containing 1% FCS, cells were stained with annexin V-FITC (Boehringer Mannheim, Mannheim, Germany) annexin V-PE (Pharmingen, San Diego, CA) and 7-AAD (Sigma España) according to the instructions of the manufacturer. Cells were analyzed on a FACSCalibur.

Coreceptor re-expression assay

Conditions for Pronase stripping and coreceptor re-expression followed the protocol developed by Suzuki et al. (24). Briefly, thymocytes were washed extensively and treated with 0.04% Pronase (Sigma España), and 100 mg/ml DNase I (Boehringer Mannheim) at 37°C for 15 min. Cells were then pelleted and incubated with fresh Pronase for 10 min at 37°C, after which the reaction was quenched with FCS. Cells were placed in overnight culture at 37°C, stained, and analyzed.

Isolation of lineage-uncommitted DP thymocytes

As described by Suzuki et al. (24), suspensions of whole thymocytes were treated with Pronase and cultured overnight at 37°C to allow coreceptor re-expression. Lineage-uncommitted DP thymocytes are defined as cells that re-express both CD4 and CD8 following this treatment. In contrast, cells that have recently committed to either CD4 or CD8 lineage re-express only either the CD4 or CD8 coreceptor (24, 25, 26). To obtain lineage-uncommitted DP thymocytes, Pronase-treated cells were stained with anti-CD4 and anti-CD8, and purified by magnetic sorting using VarioMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) in conjunction with an anti-FITC Multisort Kit and anti-PE Microbeads (Miltenyi Biotec), following the manufacturer’s instructions.

Preparation of thymic stromal cells

Fetal (20-day-old), neonatal (1-day-old), and adult thymic fragments were cultured floating on Millipore filters (8 µm pore size) in 1.35 mM 2-dGuo (Sigma España) for 7 days and trypsinized (0.25% trypsin in 0.02% EDTA, Sigma España) to form a single-cell suspension. Residual thymocytes were depleted by treatment with anti-TCR{alpha}{beta} and anti-TCR{gamma}{delta} (PharMingen), bound to sheep anti-mouse Ig-coated magnetic beads (Dynal, Oslo, Norway).

Cell lines

The generation and characterization of a stable mouse Ltk- fibroblast cell line overexpressing a full-length construct of rat Jagged1 tagged with a triple tandem repeat of an influenza virus hemagglutinin epitope (Jagged-transfected (JT) cells) was reported by Lindsell et al. (27). JT cells and parental L cells were maintained in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Harlan Sera-Lab, Leicestershire, U.K.) at 37°C in 5% CO2.

Expression of Jagged1 on JT cells was confirmed by flow cytometry after permeabilizing cells and staining with anti-HA (12CA5) mAb (Boehringer Mannheim), followed by FITC-conjugated F(ab')2 of rabbit anti-mouse IgG (Jackson ImmunoResearch).

High oxygen submersion (HOS) thymus reaggregation cultures

The basic procedures for HOS cultures have been described previously (28, 29). Fetal, neonatal, or adult thymic stromal cells and lineage-uncommitted DP thymocytes from fetal (19-day-old) or adult rat thymuses were mixed at a ratio of 1:2. In some experiments, reaggregation cultures were made mixing adult lineage-uncommitted DP cells and adult thymic stromal cells with JT cells or parental L cells, at a ratio of 2:5:5. The mixture of cells was inoculated into wells of a 96-well V-bottom plate in 0.2 ml of RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (Harlan Sera-Lab), L-Gln (2 mM), sodium pyruvate (1 mM), 2-ME (5 x 10-5 M), streptomycin (100 µg/ml), and penicillin (100 U/ml) (all obtained from Life Technologies). The plate was centrifuged at 500 x g for 3 min and placed into a plastic bag, then the air inside was replaced for a gas mixture (70% O2, 25% N2, and 5% CO2). The plastic bag was incubated at 37°C. After culture, cells were harvested and subjected to flow cytometry analysis.

In situ hybridization

Paraformaldehyde-fixed thymuses were embedded in OTC compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at -80°C until analysis. Digoxigenin-labeled antisense riboprobes for Notch1, Notch2, Notch3, Notch4, Jagged1, Jagged2, Delta1, and Delta3 were synthesized from linearized plasmid cDNA templates using T3 or T7 polymerases (Boehringer Mannheim). In situ hybridization was performed as previously described (30). Sections were photographed using a Labophot-2 microscope (Nikon, Tokyo, Japan).

Intrathymic labeling

Animals of different ages (2-h-old, and 1-, 7-, and 15-day-old) were anesthetized by hypothermia or with Metafane (Pitmann-Moore, Mundelein, IL), the chest cavity opened, and ~5 µl of solution of FITC (0.5 mg/ml; Sigma España) injected intrathymically with a 33-gauge needle. Adult rats were intrathymically injected with 20 µl of FITC (1 mg/ml). Control animals received the same amount of FITC dropped into the mediastinal cavity. The chest was then closed with sutures. After 14 h the spleen, blood, and all the lymph nodes were harvested and stained as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low CD4:CD8 cell ratios seen during fetal life originate in the thymus

In normal adult animals, CD4+ peripheral T cells are more abundant than CD8+ T cells (CD4:CD8 cell ratio = 2–4). In contrast, early in life CD8+ T cells predominate over CD4+ T cells in peripheral lymphoid organs, which results in a CD4:CD8 cell ratio of <1 (9). High CD4:CD8 cell ratios seen in adult peripheral lymphoid organs have been demonstrated to originate in the thymus (6, 7, 8). We determined whether the low peripheral CD4:CD8 cell ratios observed during fetal life also originate in the thymus. As shown in Fig. 1Go, the predominance of CD4-CD8+TcR{alpha}{beta}high cells over the CD4+CD8-TCR{alpha}{beta}high cells occurred in all the fetal lymphoid tissues analyzed, including the thymus. However, the CD4:CD8 cell ratio in the fetal thymus was slightly higher than in the fetal peripheral lymphoid organs, although it never reached the high values observed in adult lymphoid organs (Fig. 1Go). Therefore, these results reveal that the low peripheral CD4:CD8 cell ratio seen during fetal life is established in the thymus.



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  		FIGURE 1. Distinct CD4:CD8 cell ratios in peripheral T lymphocytes throughout life originate in the thymus. Thymocytes, as well as lymph node, spleen, and peripheral blood cells from adult ({square}) and 20-day-old fetal ({blacksquare}) rats were incubated with anti-CD4, anti-CD8, and anti TCR{alpha}{beta} mAbs, and the proportions of CD4+CD8-TCR{alpha}{beta}high and CD4-CD8+TCR{alpha}{beta}high cells were estimated by flow cytometry, and used to calculate the CD4:CD8 cell ratios. Data are means of three to five determinations (±SEM), including two to five rats for each age. Values used to determine the CD4:CD8 cell ratios were the following: thymus (fetal, f) 11.4:21.8, (adult, a) 40.8:16.1; peripheral blood (f) 8.9:76.7, (a) 70.9:25.3; lymph nodes (f) 5.1:73.3, (a) 70.6:26.7; spleen (f) 5.9:88.2, (a) 55.4:38.

 
CD4/CD8 lineage commitment changes throughout thymic development

The distinct CD4:CD8 cell ratios observed during thymus development could be explained by variations in the intrathymic process of CD4 vs CD8 lineage commitment. Singer and collaborators (24) have proposed that lineage commitment in CD4+CD8+ DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. They also showed that this is a highly regulated event that is only evident within the most differentiated DP subset, expressing high levels of TCR{alpha}{beta}, CD5, CD69, and bcl-2 (25). We have used the coreceptor re-expression assay described by these authors (24) to detect the coreceptor molecules that individual DP thymocytes are actively synthesizing in the fetal, neonatal, and adult thymus, and to determine the proportion of CD4 or CD8 lineage-committed DP thymocytes and then the ratio in which single positive cells are generated before being affected by the postpositive selection proliferation and survival events. Table IGo shows that a significant proportion of perinatal DP TCR{alpha}{beta}high thymocytes had selectively terminated synthesis of CD4, whereas only a few cells had terminated synthesis of CD8. From the end of the first week of postnatal life, DP TCR{alpha}{beta}high thymocytes contained increased proportions of lineage-committed CD4+ cells (Table IGo).


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  		Table I. Coreceptor re-expression assay

 
Therefore, these results suggest that DP thymocytes generated from the first wave of T cell precursors colonizing the thymic gland (and which are maturing and being selected in the thymus during the fetal period and the first days of postnatal life; Ref. 31, 32) produce low numbers of lineage-committed CD4+ cells. On the contrary, DP cells generated from the successive waves of precursor cells maturing in the thymus (beginning from the second week of postnatal life; Refs. 31, 32) give rise to a significantly higher number of lineage-committed CD4+ cells in comparison to the progeny of the first wave of T cell maturation.

The proportions of CD4+CD8- and CD4-CD8+ thymocytes are regulated by the developmental age of the thymic stroma

The differential production of CD4 and CD8 lineage-committed cells can be regulated by factors intrinsic to the differentiating thymocytes and/or to the selecting thymic stroma. To address this issue we conducted thymus reaggregation cultures, in which lineage-uncommitted CD4+CD8+ thymocytes, purified as previously reported (26), were isolated from fetal and adult thymuses (Fig. 2GoA) and reaggregated with stromal cells from dGuo-treated fetal, neonatal, and adult thymus fragments.



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  		FIGURE 2. Developmental age of the thymic stroma regulates the proportions of CD4+ and CD8+ thymocytes. A, Fetal and adult lineage uncommitted DP thymocytes were purified as described in Materials and Methods, and the expression of CD4, CD8, and TCR{alpha}{beta} was analyzed before culture with thymic stroma. B, Generation of mature TCR{alpha}{beta}high cells after culturing fetal and adult lineage uncommitted DP thymocytes with fetal, neonatal, and adult thymic stromal cells (ratio 2:1) in HOS-thymus reaggregation cultures for 3 days. Dot plots show CD4 vs CD8 expression on thymocytes. The expression of TCR{alpha}{beta} on gated CD4+CD8- and CD4-CD8+ cells is shown in the histograms. Numbers shown in black squares represent the CD4:CD8 cell ratios of the mature subsets, calculated with the percentages of total CD4+CD8-TCR{alpha}{beta}high and CD4-CD8+TcR{alpha}{beta}high thymocytes.

 
After 3 days, the culture of both fetal and adult uncommitted CD4+CD8+ thymocytes with fetal thymic stromal cells led to selective generation of CD4-CD8+ cells expressing high levels of TCR{alpha}{beta}, whereas only a few CD4+CD8- TCR{alpha}{beta}high cells weredetected in these cultures (Fig. 2GoB). In contrast, fetal and adult uncommitted DP thymocytes cultured with adult thymic stromal cells produced large numbers of CD4+CD8-TCR{alpha}{beta}high cells but very few CD4-CD8+TcR{alpha}{beta}high thymocytes (Fig. 2GoB). An intermediatecondition was observed when lineage-uncommitted DP cells were cultured with stromal cells from neonatal thymuses (Fig. 2GoB).

These data support the view that the thymic nonlymphoid environment determines the proportions of CD4+CD8- and CD4-CD8+ cells generated in the thymus, and those proportions vary according to the developmental age of the thymic stroma.

Phenotypic differences of epithelial cells during thymus development

Several authors have previously demonstrated that, among thymic stromal cell components, epithelial cells have the exclusive property of mediating positive selection (33, 34, 35), an event in which immature thymocytes commit to either the CD4+ or CD8+ T cell lineage. The surface expression of MHC molecules on the epithelial cells used for the reaggregation cultures was examined by flow cytometry to evaluate whether changes in their phenotype could account for the differential generation of CD4- and CD8-committed cells. Fig. 3Go shows that most adult and neonatal cytokeratin-positive epithelial cells expressed MHC class I and II molecules, whereas a lower proportion of the fetal thymic epithelial cells expressed these MHC Ags. In addition, the level of expression of both class I and II MHC molecules was always lower in the fetal population than in the neonatal and adult epithelial cell populations (Fig. 3Go). Therefore, these results indicate that the different proportions of CD4 and CD8 lineage-committed cells generated throughout thymus development are not a consequence of a differential expression of MHC molecules on epithelial cells because the increase in the percentage of MHC-positive cells and in the level of expression equally affect to both class I and II MHC molecules.



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  		FIGURE 3. Phenotype of the thymic epithelial cells. Fetal, neonatal, and adult thymic stromal cell suspensions were obtained by tripsinizing thymic fragments cultured with 2-dGuo for 7 days, and immunomagnetic depletion of residual thymocytes. After surface staining with different mAbs, cells were permeabilized and stained with FITC-conjugated anti-cytokeratin mAb. Histograms represent the expression of different Ags on gated cytokeratin-positive thymic epithelial cells (gray profiles). Isotype-matched irrelevant Abs were used as negative controls to define background fluorescence (white profiles). The percentages of positive cells and their mean fluoresce intensity (M) are indicated in each histogram.

 
Differential expression of Notch receptors and their ligands during thymus development

Recent reports have indicated that Notch1 plays a critical role in CD8+ T cell maturation (18, 19). To examine the possible contribution of Notch family members to the generation of different intrathymic CD4:CD8 cell ratios, we analyzed by in situ hybridization the expression of specific transcripts for Notch receptors and their ligands in the fetal, neonatal, and adult thymus.

In correlation to the higher production of CD8+ cells during fetal life, the expression of Notch1, -2, and -3 was maximal in the early fetal thymus. The intensity of the staining decreased during the first week of postnatal life in the case of Notch1, and just before birth for Notch2 and Notch3 (Fig. 4Go, Table IIGo). Likewise, the expression of Notch ligands Jagged1 and Jagged2 in the developing thymus progressively decreased from embryonic to postnatal/adult life (Fig. 4Go, Table IIGo). On the contrary, the expression of Delta-like 1, which was first detected on fetal day 18, and Delta-like 3 did not change throughout thymus development (Table IIGo).



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  		FIGURE 4. Expression of Jagged1, Jagged2, and Notch3 in fetal, perinatal, and adult thymus was examined by in situ hybridization with digoxigenin-labeled antisense riboprobes. Expression of Jagged1 (a, d, g, and j), Jagged2 (b, e, h, and k), and Notch3 (c, f, i, and l) is maximal in the fetal thymus (a–f), decreasing around birth (g–i) to become weak or absent in the adult thymus (j–l). T, thymus; H, heart; A, aorta; R, ribs; BV, blood vessels. Scale bars denote 500 µm in a–c and 50 µm in d–l.

 

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  		Table II. Expression of Notch receptors and their ligands during thymus development1

 
These data suggest that the down-regulation of the expression of some components of Notch receptor family and their ligands Jagged1 and Jagged2 could be one of the main factors governing the differential production of CD4+CD8- and CD4-CD8+ thymocytes during thymic development.

The overexpression of Jagged1 in an adult thymic environment leads to the reduction of the CD4:CD8 cell ratio

As an approach to test this hypothesis, we analyzed whether the high CD4:CD8 cell ratios obtained after culturing adult uncommitted DP thymocytes with adult thymic stromal cells could be decreased by increasing the expression of the Notch ligand, Jagged1. Jagged1 is expressed by thymic stromal cells, unlike Notch1, -2, -3, and Jagged2, which are expressed by both thymic lymphoid and stromal cell components (36, 37). We used a stable Ltk- fibroblast cell line overexpressing rat Jagged1 tagged with a triple tandem repeat of an influenza virus hemagglutinin epitope to allow immunodetection of the Jagged protein (JT cells) (27). Expression of Jagged1 was confirmed by flow cytometry (Fig. 5GoA). Our assay involved reaggregation of adult uncommitted DP thymocytes with adult thymic stromal cells and either the JT cells or the parental L cells. After 4 days in culture, a high CD4:CD8 cell ratio was observed in the reaggregation cultures with or without the parental L cell line (Fig. 5GoB). However, the presence of the JT cells produced a 2- to 3-fold decrease in the CD4:CD8 cell ratio. This reduction was due to an increase in the proportion and absolute number of CD4-CD8+TcR{alpha}{beta}high thymocytes, without affecting notably the number of CD4+CD8-TCR{alpha}{beta}high cells (Fig. 5GoB). To determine whether the increase in the proportion of CD4-CD8+TcR{alpha}{beta}high cells was due to a preferential proliferation or survival of this thymocyte subset, we analyzed the percentage of cycling and apoptotic cells in the single positive thymocyte subpopulations produced in the reaggregation cultures. As shown in Fig. 5Go, C and D, there were no significant differences in the proportions of cycling and apoptotic CD4-CD8+ cells appearing in the different reaggregation cultures.



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  		FIGURE 5. Overexpression of Jagged1 decreases the CD4:CD8 cell ratio. A, Expression of Jagged1 on JT cells was confirmed by flow cytometry after permeabilizing cells and staining with anti-HA (12CA5) mAb followed by FITC-conjugated F(ab')2 of rabbit anti-mouse IgG. Gray histogram, JT cells; white histogram, parental L cells. Dotted lines represent the background fluorescence after staining JT cells with the secondary Ab only. B, Adult lineage uncommitted DP thymocytes were reaggregated with adult thymic stromal cells, and JT cells or parental L cells (ratio 2:5:5) and cultured under HOS conditions for 4 days. Dot plots represent CD4 vs CD8 expression on gated TCR{alpha}{beta}high thymocytes. Numbers in black squares represent the CD4:CD8 cell ratios of the mature thymocyte populations. The percentage of recovery of viable lymphoid cells relative to the number of input T cells was: Control 3.7%; +L cells 4.2%; +JT cells 3.8%. C and D, Thymocytes obtained from the reaggregation cultures were stained with anti-CD4 and anti-CD8, and their DNA content was assessed by 7-AAD staining (C) or staining with annexin V and 7-AAD. Percentages of cycling (C) and apoptotic (annexin V+ 7-AAD-) (D) CD4+CD8- (gray) and CD4-CD8+ (black) cells were calculated. Data are means (±SEM) of two different experiments.

 
Thus, these results show that the differential expression of the Notch ligand, Jagged1, influences the generation of distinct CD4:CD8 cell ratios during thymus development, and this is not caused by effects on survival or proliferation of specific thymocyte subpopulations.

Intrathymic CD4:CD8 cell ratio sharply changes around birth

To determine the timing of conversion from low, fetal-like to high, adult-like CD4:CD8 cell ratios, we determined the percentages of both mature CD4+CD8-TCR{alpha}{beta}high and CD4-CD8+TcR{alpha}{beta}high thymocytes throughout thymus development.

A predominance of CD4-CD8+TcR{alpha}{beta}high thymocytes over the CD4+CD8-TCR{alpha}{beta}high subset (CD4:CD8 cell ratio = 0.3–0.6) was observed on fetal days 19–20 (Fig. 6Go, A and B). From this developmental stage until birth time, the proportion of mature CD4+CD8-TCR{alpha}{beta}high thymocytes experienced a 6-fold increase, whereas that of CD4-CD8+TcR{alpha}{beta}high thymocytes only increased 2-fold, which resulted in higher, adult-like CD4:CD8 cell ratios (CD4:CD8 cell ratio = 1.5–2) (Fig. 6Go, A and B). In the following weeks of postnatal life the proportion of CD4+CD8-TCR{alpha}{beta}high thymocytes remained virtually unchanged. The percentage of CD4-CD8+TcR{alpha}{beta}high cells gradually decreased until the third week of postnatal life, when the numbers of both mature thymocyte subsets increased again to reach the adult values (Fig. 6Go, A and B). Accordingly, during the first 2–3 wk of postnatal life CD4+CD8-TCR{alpha}{beta}high thymocytes dominated over the CD4-CD8+TcR{alpha}{beta}high cell subset (CD4:CD8 cell ratio = 3–4) to a greater degree than they do in the adult stage (CD4:CD8 cell ratio = 2–3) (Fig. 6Go, A and B).



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  		FIGURE 6. Intrathymic CD4:CD8 cell ratio changes around birth time. Thymocytes from different developmental stages were incubated with anti-CD4, anti-CD8, and anti-TCR{alpha}{beta} mAbs, and the percentages of mature CD4+CD8-TCR{alpha}{beta}high ({blacktriangleup}) and CD4-CD8+TcR{alpha}{beta}high ({circ}) cells (A), as well as the CD4:CD8 cell ratios (B), were determined by flow cytometry. Data are means of three to eight determinations (±SEM), including two to eight rats for each age.

 
Therefore, these results demonstrate that the low, fetal-like CD4:CD8 cell ratio is detected during 2–3 days of late fetal thymus, sharply changing to adult values around birth.

Differences in the proliferative rate of mature thymocytes contribute to change the CD4:CD8 cell ratio during the perinatal period

Our results showed that the intrathymic CD4:CD8 cell ratio increased from fetal to adult values in just 1–2 days. Together with the changes in the intrathymic process of lineage commitment affecting CD4+CD8+ thymocytes, other modifications involving CD4+CD8-/CD4-CD8+ thymocyte subsets could be contributing to the rapid perinatal change in the CD4:CD8 cell ratio.

A cell cycle analysis was conducted using 7-AAD in combination with anti-TCR{alpha}{beta} mAbs to analyze the proliferative status of electronically sorted CD4+CD8- and CD4-CD8+ thymocytes from different developmental stages. Remarkably, we found that on fetal day 20 the percentage of proliferating mature CD4-CD8+TcR{alpha}{beta}high thymocytes was >3 times higher than that of CD4+CD8-TCR{alpha}{beta}high thymocytes (Fig. 7GoA). However, in the following developmental stages the proportion of cycling CD4-CD8+TcR{alpha}{beta}high cells dropped, whereas the percentage of cycling mature CD4+CD8-TCR{alpha}{beta}high thymocytes notably increased (Fig. 7GoA). Throughout postnatal life, the percentages of proliferating mature CD4+ and CD8+ thymocytes showed similar decreasing kinetics to reach the low adult values after the second week of life (Fig. 7GoA).



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  		FIGURE 7. Cell proliferation and apoptosis in mature thymocyte subsets. A, Electronically sorted CD4-CD8+ and CD4+CD8- thymocytes were stained with anti-TCR{alpha}{beta} and permeabilized, and their DNA content was assessed by 7-AAD staining and flow cytometry. Percentages of CD4+CD8-TCR{alpha}{beta}high ({blacktriangleup}) and CD4-CD8+TcR{alpha}{beta}high ({circ}) thymocytes in S+G2+M phases were calculated. Data are means of three to four experiments (±SEM). B, Thymocytes were stained with anti-CD4 (or anti-CD8), anti-TCR{alpha}{beta}, and annexin V. The proportion of apoptotic annexin V-positive cells was calculated in the CD8-TCR{alpha}{beta}high (which represent mature CD4+CD8- thymocytes) and CD4-TCR{alpha}{beta}high (which represent mature CD4-CD8+ thymocytes) cell subsets. Total thymocytes cultured overnight were used as a positive control for annexin V binding. Data are representative of three independent experiments.

 
These results correlate with the predominance of mature CD4-CD8+TcR{alpha}{beta}high thymocytes on fetal day 20, and the large increase in the CD4+CD8-TCR{alpha}{beta}high cell subset occurring in the last days of embryonic life. Therefore, modifications in the proliferative status of mature thymocytes could partially account for the change in the intrathymic CD4:CD8 cell ratio occurring in the perinatal period.

Cell death does not influence the perinatal change of CD4:CD8 cell ratio

Annexin V binding (38) was used to determine whether a differential apoptosis rate of the mature thymocyte subsets in the fetal and adult thymus could also contribute to generate the distinct CD4:CD8 cell ratios found. However, low levels of apoptotic cells (<2%) were detected among mature CD4+ and CD8+ thymocytes throughout thymus development (Fig. 7GoB), indicating that apoptosis does not influence the perinatal change of the intrathymic CD4:CD8 cell ratio.

The perinatal change of the CD4:CD8 cell ratio is also promoted by differences in thymocyte emigration to peripheral lymphoid organs

A differential emigration rate of mature thymocyte subsets to the periphery could also contribute to the rapid change in the intrathymic CD4:CD8 cell ratio. Thus, we analyzed the proportion of CD4+CD8- and CD4-CD8+ cells appearing in the TCR{alpha}{beta}+ cell population from mesenteric and peripheral lymph nodes, spleen, and peripheral blood. This is a reliable approach to study thymocyte emigration during the perinatal period, as we found that colonization of peripheral lymphoid organs by TCR{alpha}{beta}+ cells started on days 20–21 of fetal life. From this we conclude that most of the peripheral T cells found in the perinatal period have been recently derived from the thymus.

Interestingly, in the last days of fetal life CD4-CD8+TcR{alpha}{beta}+ cells outnumbered CD4+CD8-TCR{alpha}{beta}+ cells in all the peripheral lymphoid organs analyzed (Table IIIGo), leading to depressed CD4:CD8 cell ratios. These ratios were lower than the intrathymic ratios found in the same fetal period. However, after birth CD4-CD8+TcR{alpha}{beta}+ cells were rapidly overtaken in proportion and number by CD4+CD8-TCR{alpha}{beta}+ cells, increasing the CD4:CD8 cell ratio that reached adult values by the end of the first week of postnatal life (Table IIIGo).


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  		Table III. Phenotype of TCR{alpha}{beta}high cell subpopulations appearing in rat peripheral lymphoid organs1

 
To determine whether the higher numbers of peripheral CD4-CD8+TcR{alpha}{beta}+ cells found around birth corresponded to extrathymically derived T cells, we further analyzed the expression of CD8{beta} in this T cell subpopulation. Extrathymically originated T cells were identified through expression of the CD8{alpha}{alpha} homodimer rather than the CD8{alpha}{beta} heterodimer (39). Fig. 8GoA shows that on fetal day 21 the large majority of CD4- TCR{alpha}{beta}+ cells expressed both CD8{alpha} and CD8{beta} molecules in all the peripheral lymphoid organs analyzed, confirming the thymic origin of this cell subset. These results were consistently obtained in all developmental stages studied (data not shown).



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  		FIGURE 8. Thymocyte emigration influences the CD4:CD8 cell ratio. A, Mesenteric lymph node, spleen, and peripheral blood cells from 21-day-old fetuses were triple-labeled for flow cytometry analysis, and the expression of CD4, CD8{alpha}, CD8{beta}, and TCR{alpha}{beta} was examined. Histograms show the expression of CD8{alpha} and CD8{beta} in CD4-TCR{alpha}{beta}+ cells gated in CD4/TCR{alpha}{beta} dot plots. B, Mesenteric lymph node cells from different developmental stages were stained with either anti-CD4 or anti-CD8, and anti-TCR{alpha}{beta} mAbs, permeabilized, and incubated with 7-AAD. The proportions of CD8-TCR{alpha}{beta}high (which represent mature CD4+CD8- lymphocytes) and CD4-TCR{alpha}{beta}high (which represent mature CD4-CD8+ lymphocytes) cells in S+G2+M phases were calculated. Data are means of two to four experiments (±SEM). Similar results were obtained using peripheral lymph node cells or splenocytes.

 
We also determined the cell cycle status of peripheral CD4+CD8-TCR{alpha}{beta}+ and CD4-CD8+TcR{alpha}{beta}+ cells to know whether CD8+ T cells were selectively expanding in the periphery during the perinatal period. Both peripheral TCR{alpha}{beta}+ cell populations contained a high proportion of cycling cells in the last days of fetal life, which gradually diminished until reaching the minimal values found in adults (Fig. 8GoB). However, no significant differences were found in the proportion of cycling CD4+CD8- and CD4-CD8+ T cells in any of the stages studied (Fig. 8GoB).

Taken together our findings suggest that emigration of CD4-CD8+ thymocytes is favored in the perinatal thymus. This was supported by results obtained after analyzing neonatal recent thymic emigrants (RTE). For this purpose, 2-h-old as well as 1-, 7-, 15-day-old, and adult rats were intrathymically injected with FITC and 14 h later, the lymph nodes, spleen, and peripheral blood were analyzed for FITC-labeled cells. Table IVGo shows that RTE leaving the thymus during the first 24–48 h of extrauterine life were enriched in CD4-CD8+ cells when compared with RTE populations emigrating to the periphery in 1- to 2-wk-old and adult animals. Accordingly, CD4:CD8 cell ratios of RTE in all peripheral lymphoid organs analyzed were lower soon after birth, and gradually increased with age (Table IVGo).


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  		Table IV. Phenotype of the recent thymic emigrants in peripheral lymphoid organs from postnatal and adult rats1

 
Therefore, we show that the preferential emigration of CD8+ thymocytes during the last days of embryonic development and the first days of postnatal life is another factor that promotes the change of the intrathymic CD4:CD8 cell ratio in the perinatal period, and largely accounts for the depressed CD4:CD8 cell ratios found in the periphery around birth.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In adult life, the proportion of CD4+ cells among T lymphocytes in peripheral lymphoid organs is higher than that of CD8+ cells, whereas early in development CD4+ cells are underrepresented among peripheral T cells (9). Accordingly, the ratio of CD4+ to CD8+ T lymphocytes is significantly higher in adult animals (CD4:CD8 cell ratio = 2–4) than in young animals (CD4:CD8 cell ratio <1).

In this study we demonstrate that the low CD4:CD8 cell ratio observed among peripheral T cells during fetal life originates in the thymus. In the same way, our data and those previously reported (6, 7, 8) show that the high CD4:CD8 cell ratios seen in adult peripheral lymphoid organs also have an intrathymic origin. Our results indicate also that these distinct intrathymic CD4:CD8 cell ratios can be generated by variations in the intrathymic processof CD4 vs CD8 lineage commitment. The perinatal CD4+CD8+TCR{alpha}{beta}high thymocyte subset contains a significantly lower number of lineage-committed CD4+ cells than the adult CD4+CD8+TCR{alpha}{beta}high cell subpopulation. We found that this differential production of CD4+ and CD8+ cells is regulated by the developmental age of the thymic stroma. The culture of fetal and adult uncommitted DP thymocytes with fetal thymic stromal cells gives rise to low CD4:CD8 cell ratios, whereas the culture with adult thymic stroma leads to high CD4:CD8 cell ratios, irrespective of the age of the DP thymocyte subset. Previously, Adkins (10) pointed out that the differentiation potential of fetal CD4-CD8- thymocytes differed from that of adult CD4-CD8- thymic precursors. Unlike adult thymic precursors, fetal thymic progenitors gave rise to reduced numbers of CD4+ peripheral T cells in chimeric animals (CD4:CD8 cell ratio <1). This relative under-representation of CD4+ peripheral T cells was seen 4–16 wk after either intrathymic injection of suspensions of fetal CD4-CD8- thymocytes into adult thymuses, or transplantation of fetal thymic lobes under the kidney capsule of adult host animals. Then, fetal thymic precursors seemed to be committed to a relative underproduction of CD4+ peripheral T cells, regardless of the developmental age of the thymic stromal environment. However, in a previous report, Guidos et al. (40) showed that fetal and adult precursors, intrathymically injected in adult thymuses, produced similar proportions of CD4+ and CD8+ cells (CD4:CD8 cell ratio 2–3) within the thymus as well as in the peripheral organs 2 wk postinjection. The decline in the CD4:CD8 cell ratio observed in the following weeks was thought to be due to differences in the life span and/or proliferative capacity of peripheral T cells derived from fetal vs adult precursor cells. Thus, in agreement with our results, the developmental age of the thymic stromal environment seems to be the key regulatory element involved in the generation of distinct proportions of CD4+ and CD8+ thymocytes.

Among thymic stromal cell components, epithelial cells are unique in their ability to mediate the maturation of CD4+CD8+ thymocytes into CD4+ or CD8+ T cells (33, 34, 35). In this process the proportions of class I and II MHC-expressing epithelial cells have been shown to be able to influence the proportion of CD8+ and CD4+ thymocytes (41). The surface phenotypic analysis of fetal, neonatal, and adult thymic epithelial cells reveals a slight increase in the proportion of both class I and II MHC-expressing cells throughout thymus development. However, these differences are insufficient to explain the generation of distinct CD4:CD8 cell ratios throughout life. The level of expression of both class I and II MHC molecules notably increases from fetal to adult developmental stages, but because these differences are very similar for both MHC molecules, they cannot account for the differential generation of CD4+ and CD8+ thymocytes either. Instead, they could be more related to a maturational process, because similar differences are detected in the level of expression of other molecules such as CD54 and CD48 (our unpublished observations).

Signals from receptors other than those recognizing MHC molecules have been proposed to influence CD4/CD8 lineage commitment. Recently, Notch signaling, known to play a pivotal role in cell-fate determination in many tissues, has been implicated in the maturation of CD8+ but not CD4+ T cells (18, 19). We analyzed the expression of Notch receptors and their ligands, Jagged and Delta, in fetal, neonatal, and adult thymus. It has been reported that Notch1, -2, and -3, and Jagged2 are expressed by both thymic lymphoid and stromal cell components, whereas Jagged1 and Delta-like 1 and 3 molecules seem to be mostly expressed by epithelial cells (36, 37). We show that the expression of Notch1 to -3 and their ligands Jagged1 and -2, but not Delta-like 1 and 3 molecules, is maximal during fetal life, and decreases in the neonatal/adult thymus. Similar results have been reported for Notch1 (21) and Jagged1 (36) in the developing mouse thymus. This down-regulation of the expression of Notch receptors and Jagged ligands occurring from fetal to adult stages correlates to the increasing (decreasing) proportions of CD4+(CD8+) cells generated after culturing uncommitted DP thymocytes with fetal, neonatal, and adult thymic stromal cells. The involvement of Notch signaling in the generation of distinct intrathymic CD4:CD8 cell ratios throughout life was confirmed after reaggregation of adult uncommitted CD4+CD8+ thymocytes and adult stromal cells with L cells stably overexpressing rat Jagged1. A 2- to 3-fold reduction in the CD4:CD8 cell ratio is observed in these cultures, in comparison to those lacking the JT cells or including the parental L cells. This reduction is attributed to an increase in the number of CD4-CD8+TCR{alpha}{beta}high thymocytes, whereas that of CD4+CD8-TCR{alpha}{beta}high cells hardly changes. These data are in contrast to those describing that the expression of constitutively active Notch1 in thymocytes resulted in the development of excess CD4-CD8+ thymocytes at the expense of CD4+CD8- thymocytes (18). More recently, and in line with our findings, Yasutomo et al. (19) demonstrated that the blockade of Notch1 activity did interfere with the development of CD8+, but not CD4+, T cells. Likewise, Deftos et al. (42) reported that Notch1 signaling promotes the maturation of DP thymocytes into CD4+ and, in a higher extent, CD8+ thymocytes. We also demonstrate that these higher numbers of CD4-CD8+ thymocytes are not due to a preferential survival or proliferation of this thymocyte subset, which agrees with previous findings from Robey et al. (18, 43). The way in which Notch promotes the differentiation of CD8+ T cells has not been definitively established. Although some authors report that Notch would exert its effect directly on lineage commitment (43), others point out that the maturation/survival step of DP cells occurring after lineage commitment, rather than the actual choice between CD4 and CD8 fates, might represent the Notch signaling-dependent step (19, 42).

We also demonstrate that low intrathymic CD4:CD8 cell ratios are detected during the last days of fetal life, when the first mature thymocytes are generated, and that just before birth the CD4:CD8 cell ratio sharply changes from fetal to adult values. The changes in the intrathymic process of CD4/CD8 lineage commitment that we describe in this report account for the generation of the distinct fetal and adult CD4:CD8 cell ratios, as well as the maintenance of the high CD4:CD8 cell ratio throughout adult life. However, those changes cannot explain the rapid perinatal change in CD4:CD8 cell ratio because they occur more gradually, as suggested by the intermediate condition, between fetal and adult, observed with the reaggregation cultures using neonatal thymic stromal cells. Therefore, other mechanisms, such as variations in the proliferation, survival, or thymic emigration of the mature thymocytes, could be operating in the perinatal period to modify the representation of these thymocyte subsets. We demonstrate that the proliferative rate of mature CD8+ thymocytes decreases in the last days of fetal life, whereas the proportion of cycling cells in the CD4+ thymocyte subset undergoes a 2-fold increase in the same period. An increased responsiveness to IL-2 and IL-7 has been suggested to be involved in the enhanced proliferative rate exhibited by the perinatal CD4+ thymocyte subset (44, 45). We also found that mature CD8+ thymocytes preferentially leave the thymus during the perinatal period. This is supported by several lines of evidence: 1) thymus-derived CD4-CD8+ T cells predominate in the peripheral TCR{alpha}{beta}+ cell populations during the last days of fetal life, when the peripheral lymphoid organs are colonized by the first T lymphocytes; 2) RTE leaving the thymus during the first 24–48 h of extrauterine life are enriched for mature CD8+ cells when compared with RTE populations emigrating to the periphery in older animals. Moreover, a comparative phenotypic study on adult and perinatal mature CD8+ thymocytes shows that a larger proportion of perinatal CD8+ cells express higher levels of adhesion molecules (data not shown), which might explain the selective emigration of this mature thymocyte subset in fetal and neonatal animals.

In conclusion, our results indicate that thymic epithelial cells can regulate the generation of distinct CD4:CD8 cell ratios during thymus development through Jagged1-induced Notch signaling. Because Jagged1 is not the only Notch ligand expressed by the thymic epithelial cells, it is also possible that other Notch ligands could also influence the generation of the intrathymic CD4:CD8 cell ratios. Our data also provide evidence that mechanisms such as selective proliferation and emigration from the thymus of some mature thymocyte subsets contribute to the rapid change of the intrathymic CD4:CD8 cell ratio from fetal to adult values in the perinatal period.


   Acknowledgments
 
We thank Adriana Bonomo for help with the intrathymic injections, and Tessa Crompton for critically reading this manuscript.


   Footnotes
 
1 This work was supported by grants from the Ministerio de Educación y Cultura (PB97-0332), Fondo de Investigaciones Sanitarias (FIS 98/0041) Comunidad de Madrid (08.3/0014/1997 and 08.3/0027/1998), and Ministerio de Ciencia y Tecnología (PM99-0060). E.J. is a fellow of the Ministerio de Educación y Cultura. Back

2 Address correspondence and reprint requests to Dr. Alberto Varas, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain. Back

3 Abbreviations used in this paper: DP, double positive; HOS, high oxygen submersion; JT, Jagged-transfected; RTE, recent thymic emigrants; 7-AAD, 7-amino actinomycin D. Back

Received for publication September 29, 2000. Accepted for publication March 5, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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