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Blood, Vol. 94 No. 10 (November 15), 1999: pp. 3491-3498

beta -Selection Is Associated With the Onset of CD8beta Chain Expression on CD4+CD8&b.alpha;&b.alpha;+ Pre-T Cells During Human Intrathymic Development

By Yolanda R. Carrasco, César Trigueros, Almudena R. Ramiro, Virginia G. de Yébenes, and María L. Toribio

From the Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta  locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha ) chain. Such a critical developmental checkpoint, known as beta -selection, results in progression from CD4- CD8- double negative (DN) to CD4+ CD8+ double positive (DP) TCRalpha beta - thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4+ CD8- intermediate stage. Here we show that the CD4+ CD8- to CD4+ CD8+ transition involves the sequential acquisition of the alpha and beta  chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha , but not CD8beta , is expressed in vivo in a minor subset of DP TCRalpha beta - thymocytes, referred to as CD4+ CD8alpha alpha + pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta ic). In contrast, expression of CD8alpha beta heterodimers was selectively found on DP TCRalpha beta - thymocytes that express TCRbeta ic and are enriched for cycling cells. Interestingly, CD4+ CD8alpha alpha + pre-T cells are shown to be functional intermediates between CD4+ CD8- TCRbeta ic- and CD4+ CD8alpha beta + TCRbeta ic+ thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta ic expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4+ CD8alpha alpha + to CD4+ CD8alpha beta + transition marks the key control point of pre-TCR-mediated beta -selection in human T-cell development.
© 1999 by The American Society of Hematology.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BONE MARROW-DERIVED lymphoid progenitors seed the postnatal thymus in which they undergo the sequential rearrangement of the beta  and alpha  T-cell receptor (TCR) genes to finally generate mature T lymphocytes bearing an alpha beta TCR.1-3 Early during the intrathymic developmental process, pre-T cells that have succeeded in productive rearrangements at the TCRbeta locus are rescued from apoptotic cell death and selected for further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha ) chain and associated with CD3.4-7 Expression of this pre-TCR complex promotes at the same time a cell-cycle transition that results in the expansion of those thymocytes expressing a functional TCRbeta chain, a process that has been termed beta -selection.7-9

In both mice and humans, such a critical developmental checkpoint is associated with the acquisition of the CD4+ CD8+ double-positive (DP) phenotype, which is then followed by a first round of TCRalpha gene rearrangement and expression.3,7-10 Differences exist, however, in the developmental timing of appearance of CD4 and CD8 in both species. Progression to the DP compartment occurs in mice through the CD44- CD25+ stage of double-negative (DN; CD4- CD8-) thymocytes via CD44- CD25- DN thymocytes.8,9 The latter subset, although currently referred to as the last DN stage, includes cells that are already transcribing and expressing low (barely detectable) levels of both CD4 and CD8, so that they spontaneously differentiate in vitro to the DP stage within 24 hours.11-13 Isolated CD44- CD25+ DN cells, in contrast, are unable to generate CD4+ CD8+ DP cells in vitro13; however, they can acquire the CD8alpha chain in response to certain combinations of cytokines,14 thus expressing the CD8alpha alpha homodimeric form. Interestingly, such CD8alpha alpha + cells remain negative for CD3 and CD4 in vitro, but can differentiate into conventional CD4+ CD8alpha beta + DP thymocytes expressing the CD3-associated alpha beta TCR under the influence of the thymic microenvironment.15 In contrast to mice, developing DN thymocytes in humans acquire CD4 before CD8 and, therefore, the DN-to-DP transition occurs in humans via CD3- CD4+ CD8- intermediates.16 These thymocytes, similarly to their mouse CD44- CD25+ DN counterparts, differentiate in vitro into cells expressing exclusively the CD8alpha alpha homodimer17 but generate common CD4+ CD8alpha beta + DP thymocytes with the mature CD3-TCRalpha beta complex in vivo.16

Current data on the physiologic expression of CD8 either as an alpha alpha homodimer or as an alpha beta heterodimer on distinct CD8+ cell types support the notion that CD8alpha alpha + T cells, such as intestinal intraepithelial lymphocytes (IEL), can be generated by an extrathymic maturation pathway independent of CD8beta expression, whereas induction of CD8beta and, hence, the generation of CD8alpha beta + cells is thymus-dependent.18,19 This is further supported by functional studies showing that the CD8beta polypeptide is critically involved in the maturation of CD8-lineage cells inside the thymus, so that both positive and negative selection of major histocompatibility complex (MHC) class-I-restricted T cells is impaired in CD8beta -/- mice.19-22 All these data concur with the fact that the CD8alpha alpha + phenotype is prominent among CD8+ T cells from athymic mice and rats, whereas normal thymocytes and CD8+ T cells from euthymic animals are virtually all CD8alpha beta +.15,19,23 Nonetheless, CD8alpha alpha + cells have been reported to exist physiologically, although in a very low proportion, in the human thymus,17 raising the possibility that they constitute an important intermediate in the pathway of generation of thymus-derived T cells in humans. Supporting this notion, results from a very recent study by Blom et al24 have shown that such CD8alpha alpha + thymocytes, which coexpress surface CD4 but still lack a mature alpha beta TCR,7 have extensive TCRbeta gene rearrangements. However, functional studies on the developmental potential and precursor-product relationships of isolated CD4+ CD8alpha alpha + thymocytes are still lacking, precluding a better understanding of the physiological relevance of this particular cell subset in the context of thymocyte development and beta -selection in humans.

Here we show that CD4+ CD8alpha alpha + human thymocytes are actually functional intermediates between CD4+ CD8- and CD4+ CD8alpha beta + thymocytes. More importantly, evidence is provided that onset of CD8beta expression and beta -selection are coincident events associated with the acquisition of CD3 and pTalpha chain on the cell surface during intrathymic development in humans. These results suggest that the pre-TCR-mediated check-point of beta -selection is placed in humans at the CD4+ CD8alpha alpha + to CD4+ CD8alpha beta + transition.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of thymocyte subsets.   Postnatal thymus samples were removed during corrective cardiac surgery of patients aged 1 month to 3 years. Thymocyte suspensions obtained by Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation were fractionated on stepwise Percoll (Pharmacia, LKB, Uppsala, Sweden) density gradients as previously described.25 Thymocytes recovered from the 1.068 and 1.08 density layers, referred to as large and small thymocytes, respectively, were depleted (>99% purity) of mature T, B, NK, and myeloid cells (Lin- cells) by immunomagnetic sorting as described elsewhere.10 Thymocytes coexpressing CD4 and CD8 (DP) were then magnetically sorted from the remaining large and small Lin- cell pool with anti-CD8-coated magnetic beads (Dynabeads, Dynal Corp, Oslo, Norway), whereas CD3- CD4+ CD8- thymocytes were sorted from the CD8-depleted pool of large cells by treatment with anti-CD4-coated magnetic beads (Dynal).10

Small DP thymocytes thus isolated were all negative for TCRalpha beta and CD3 expression, and will be hereafter referred to as small DP CD3- thymocytes.10 In contrast, large DP thymocytes included CD3- and CD3low pTalpha + thymocytes. Both subsets were independently isolated from the whole pool of large DP TCRalpha beta - thymocytes as described previously.10 These cells will be referred to as large DP CD3- and large DP CD3low thymocytes, respectively. Large DP CD3- thymocytes were fractionated into CD8alpha alpha + and CD8alpha beta + cells by cell sorting as described below.

Flow cytometry and cell sorting.   Directly labeled monoclonal antibodies (MoAb) against the following antigens were used: CD3 (Leu4-PE), CD8alpha (Leu2a-fluorescein isothiocyanate [FITC]), and CD5 (Leu1-FITC) from Becton Dickinson & Co, San José, CA; CD4 (CD4-PE-Cy5), CD44 (CD44-FITC), CD69 (CD69-FITC), and CD3 (CD3-PE-Cy5) from Caltag Laboratories, South San Francisco, CA; CD71 (T9-FITC) from Coulter Corp, Hialeah, FL; and CD28 (CD28-FITC) from Serotec Ltd, Oxford, UK. Unlabeled MoAb against CD8beta (2ST8-5H7, kindly provided by Dr E.L. Reinherz, Dana-Farber Cancer Institute, Boston, MA)26 as well as MoAb recognizing monomorphic determinants of TCRalpha beta (BMA031, generously provided by Dr R. Kurrle, Behringwerke AG, Marburg, Germany),27 were used in combination with FITC- or PE-coupled goat-antimouse F(ab)'2 immunoglobulin (Ig) (Caltag). Either unlabeled or directly labeled isotype-matched irrelevant MoAb (Caltag) were used as negative controls. For detection of cytoplasmic TCRbeta , cells were treated with 0.5% saponin (Sigma, St Louis, MO), incubated with the anti-TCRbeta chain beta F1 MoAb28 (generously provided by Dr M. Brenner, Brigham and Women's Hospital, Boston, MA) and labeled with PE- or PE-Cy5-coupled goat antimouse IgG1 (Caltag). To define background fluorescence, cells were sequentially treated with a nonreactive mouse IgG1 MoAb plus PE- or PE-Cy5-conjugated goat antimouse IgG1. Surface expression of pTalpha chain was determined by sequential staining with a rabbit polyclonal antibody (ED-1) previously described,10 and FITC-conjugated goat antirabbit F(ab)'2 Ig (Southern Biotechnology Associates, Inc, Birmingham, AL). Preimmune rabbit serum was used as negative control. Two- or three-color staining was performed as described elsewhere.25 Stained cells were analyzed in an EPICS XL flow cytometer (Coulter Electronics Inc, Hialeah, FL). Cell cycle analyses were performed by flow cytometry using a doublet discrimination function in cells treated with 0.05% digitonin (Sigma), washed, and stained with 50 µg/mL of propidium iodide (PI) (Sigma), as described elsewhere.10 Cell sorting of CD8alpha alpha + and CD8alpha beta + cells was performed in an EPICS Elite Cell Sorter (Coulter Electronics, Inc) on isolated large DP CD3- thymocytes after labeling with anti-CD8beta plus PE-labeled goat antimouse IgG2a. An irrelevant IgG2a mouse MoAb was used as negative control. Sorted cells were greater than 98% pure as determined by post-sort analysis.

Western blot analysis.   Cells were lysed for 30 minutes at 4°C in lysis buffer containing 0.5% Deoxicholate, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 50 mmol/L NaF, 1 mmol/L Na3VO4 (Sigma Chemical Co), 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0) (Merck, Darmstadt, Germany), 1 mmol/L PMSF, and 1 µg/mL each of leupeptin, pepstatin A, and aprotinin (Sigma). The protein concentration was determined by micro-bicinchoninic acid (BCA; Pierce, Rockford, IL) assay. Defined quantities (~7 µg/lane) were electrophoreses in 7% SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. Western blotting was performed as previously described.25 Blots were incubated with either the anticyclin B (Transduction Laboratories, Lexington, KY), the anti-RAG1, the anti-RAG2 (Pharmingen, San Diego, CA), or the anti-beta -tubulin (Amersham International) mouse MoAb, or with a polyclonal rabbit anticyclin A antibody, or a polyclonal goat anti-Retinoblastoma (anti-Rb) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), as primary reagents. Specific signals were shown with either horseradish perioxidase (HRPO)-labeled polyclonal sheep antimouse Igs, or HRPO-labeled polyclonal goat antirabbit Igs (Amersham International) or HRPO-labeled polyclonal rabbit antigoat Igs (Jackson Immunoresearch, West Grove, PA), respectively, and an enhanced chemiluminescence (ECL)-detection kit (Amersham International).

Hybrid human/mouse fetal thymic organ cultures.   The in vitro generation of mature TCRalpha beta + human T cells was analyzed using a modification of the previously described hybrid human/mouse fetal thymic organ culture (hu/mo FTOC).29 Briefly, thymuses removed from 15-day-old embryos of Swiss mice were precultured for 5 to 6 days in the presence of 1.35 mmol/L dGuo (Sigma). Afterwards, the thymic lobes were washed and cocultured in hanging drops in Terasaki plates (Nunc, Inc, Roskilde, Denmark) with either CD8alpha alpha + or CD4+ (3 × 104 to 105 cells/lobe) human thymocytes. After 2 days, lobes were transferred to Millipore filters (Millipore Corp, Bedford, MA), that were layered over gelfoam rafts and cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 2% human AB serum and 5% fetal calf serum (FCS; Gibco BRL, Paisley, Scotland). Surface staining of human cells was performed at the indicated culture periods, and flow cytometric analyses (FCA) were then performed on electronically gated CD45+ human cells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of CD3- CD4+ CD8alpha +beta - DP cells in the human thymus.   We have recently reported that surface CD3 expression and cell size define three subsets of human TCRalpha beta - thymocytes, namely large CD3low, large CD3-, and small CD3-, which represent distinct pre-T-cell developmental stages.10 All three pre-T-cell types expressed surface CD4 and were reactive with conventional anti-CD8 antibodies (directed against an epitope on the CD8alpha molecule), so that they were characterized as DP thymocytes.10 However, as shown in Fig 1A, analysis on the correlated expression of CD8alpha and CD8beta , performed with the anti-CD8beta MoAb 2ST8-5H7,26 showed that large CD3low and small CD3- pre-T cells were homogeneously CD8alpha +beta +, whereas a significant proportion (32 ± 4%) of large CD3- pre-T cells expressed CD8alpha but lacked CD8beta chain. Such a differential expression of the alpha  and beta  chains of CD8 defines two distinct subsets of large CD3- pre-T cells in which CD8 may be expressed either as a CD8alpha alpha homodimer or as a CD8alpha beta heterodimer,26 so that they will hereafter be referred to as either CD8alpha alpha + or CD8alpha beta +, respectively. Both cell subsets could be independently isolated by cell sorting, and were then compared for their developmental status by analyzing the intracytoplasmic expression of TCRbeta chain (TCRbeta ic). As shown in Fig 1B, a clear phenotypic pattern was obtained: TCRbeta ic expression was not detectable in CD8alpha alpha + thymocytes, whereas essentially all CD8alpha beta + pre-T cells coexpressed TCRbeta ic. These results confirm our previous data showing a bimodal distribution of TCRbeta ic within the pool of large CD3- pre-T cells as a whole10 but, in addition, they suggest that expression of TCRbeta ic parallels acquisition of the CD8alpha beta + phenotype.


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Fig 1. Differential expression of CD8alpha and CD8beta chains on distinct subsets of human pre-T cells: Intracytoplasmic TCRbeta expression and cell-cycle progression are associated with CD8beta chain expression. (A) Large CD3-, large CD3low pTalpha +, and small CD3- pre-T cells, isolated as described in Materials and Methods, were analyzed by two-color flow cytometry for CD8alpha versus CD8beta expression. (B) Large CD3- CD4+ CD8+ (DP) pre-T cells were fractionated by cell sorting into CD8alpha +beta -(top panels) and CD8alpha +beta + (bottom panels) cells after labeling with the 2ST8-5H7 anti-CD8beta MoAb plus PE-coupled goat antimouse IgG2a. Reanalysis of surface CD8beta expression postsorting is shown (shaded histograms). Sorted cells were then analyzed by flow cytometry for intracytoplasmic TCRbeta chain (TCRbeta ic) expression (shaded histograms) and DNA content. Cytoplasmic background fluorescence was determined on sorted cells stained with a nonreactive mouse IgG1 MoAb plus PE-Cy5-coupled goat antimouse IgG1 (unshaded histograms). Percentages of cycling cells (in S and G2/M phases) are indicated. Results are representative of four independent experiments.

Because TCRbeta ic expression is currently envisaged as a marker of beta -selection, we concluded that the CD8alpha alpha + and the CD8alpha beta + subsets could represent two distinct pre-T-cell developmental stages placed on either side of the beta -selection process. This was confirmed by FCA showing that both populations differed dramatically in their cell cycle status. As expected of beta -selected thymocytes, CD8alpha beta + pre-T cells featured a high proportion (up to 55%) of cycling cells, whereas essentially all (>90%) CD8alpha alpha + thymocytes were "unselected" cells arrested in the G0/G1 phases of the cell cycle (Fig 1B).

The distinct developmental status of the CD8alpha alpha + and CD8alpha beta + cell subsets of large CD3- pre-T cells, prompted us to examine in more detail their phenotypic profiles, as compared with that of large cycling CD3low pre-T cells, previously shown to represent the particular stage of beta -selected thymocytes at which the CD3-associated pre-TCR is expressed in vivo.10 Regardless of CD3 expression, the two subsets (CD3- and CD3low) of beta -selected CD8alpha beta + TCRbeta ic+ thymocytes were homogeneously positive for the expression of CD44, whereas CD8alpha alpha + pre-T cells were CD44- (Fig 2). In addition, all three pre-T-cell types displayed surface CD4, but levels of expression were consistently lower in the latter. Expression levels of activation markers such as CD71 (transferrin receptor) and, to a lesser extent, CD28 were also significantly lower in CD8alpha alpha + pre-T cells, whereas CD5 and CD69 were similarly expressed in the three populations (Fig 2).


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Fig 2. Cell-surface phenotype of individual subsets of CD8alpha alpha +and CD8alpha beta + human large pre-T cells. Large CD3- DP pre-T cells shown in Fig 1 were stained with directly labeled MoAb against the indicated cell-surface molecules. FCA was performed on electronically gated CD8alpha +beta - (shaded areas) or CD8alpha +beta + (unshaded areas, bold line) cells. Large CD3low pTalpha + pre-T cells, shown to be homogeneously CD8alpha +beta + (see Fig 1), were included in the study for comparison (unshaded areas, thin line). Background values (vertical lines) were determined with isotype-matched irrelevant MoAb. A representative analysis out of three independent experiments is shown.

CD4+ CD8alpha alpha + pre-T cells are the immediate precursors of CD4+ CD8alpha beta + pre-T cells that coexpress surface pTalpha and CD3.   To provide direct evidence that CD4+ CD8alpha alpha + thymocytes do in fact represent a minor, but physiologically relevant, population of pre-T-cell intermediates, they were next examined for their developmental potential in a hybrid hu/mo FTOC system. Highly purified CD4+ CD8alpha alpha + thymocytes (>98% pure after cell sorting) were consistently found to give rise to conventional DP cells coexpressing CD4 and the CD8alpha beta heterodimer at early periods of culture (20% by day 5 in this experiment; Fig 3A, upper panel). Of notice, essentially all CD4+ CD8alpha beta + progeny (>95%) generated by this time in different experiments had acquired low surface CD3 with minimal differentiation (<2%) into TCRalpha beta + cells (Fig 3A, lower panel). Interestingly, such CD3low TCRalpha beta - CD4+ CD8alpha beta + pre-T cells (20% of total cells recovered by day 5) were all positive for the expression of TCRbeta ic and, more importantly, they displayed low surface levels of the pTalpha chain (Fig 3B), as assessed with a polyclonal anti-pTalpha antibody.10


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Fig 3. Phenotypic analysis of the cellular progeny generated after differentiation of CD4+ CD8alpha alpha + human pre-T cells in FTOC. (A) CD4+ CD8alpha alpha + pre-T cells, isolated by cell sorting as described in Materials and Methods, were cultured in a hybrid hu/mo FTOC and analyzed after 5 and 14 days for the expression of CD8alpha , CD8beta , TCRalpha beta , and CD3. Analysis of TCRalpha beta versus CD3 was performed by three-color flow cytometry after electronic gating on the CD8beta + progeny. (B) Intracytoplasmic TCRbeta (TCRbeta ic) and surface pTalpha expression (shaded areas) was analyzed after gating on the CD3low progeny recovered by day 5. Background fluorescence was determined with isotype-matched irrelevant MoAb and with a rabbit preimmune serum. A representative experiment out of three is shown.

We have previously shown that CD4+ CD8alpha beta + pre-T cells with this particular CD3low pTalpha + phenotype are functional progenitors of common DP thymocytes that already express surface alpha beta TCR.25 Accordingly, CD4+ CD8alpha beta + cells generated in the thymic lobes increased in numbers throughout culture (up to 9- to 10-fold in 6 to 7 days) to become the major cell subset (>95%) by day 14, and acquired simultaneously intermediate to high levels of both CD3 and the mature alpha beta TCR (50% of total cells recovered by day 14 in the experiment shown in Fig 3A). These results provide evidence that CD4+ CD8alpha alpha + thymocytes represent the immediate precursors of the first intrathymic cells with a conventional CD4+ CD8alpha beta + DP phenotype. Moreover, they indicate that the developmental onset of CD8beta chain expression is closely associated with the beta -selection process and parallels the acquisition of a surface pre-TCR.

CD4+ CD8alpha alpha + pre-T cells are functional intermediates between CD4+ CD8- progenitors and CD4+ CD8alpha beta + pre-T cells.   It is currently believed that CD4+ CD8- thymocytes that still lack the CD3-TCRalpha beta are the immediate precursors of DP thymocytes in the human thymus.16 However, we show in this study that CD4+ CD8alpha alpha + pre-T cells are efficient progenitors of CD4+ CD8alpha beta + DP thymocytes, suggesting that they represent very transient intermediates between CD4+ CD8- and CD4+ CD8alpha beta + cells in vivo. To seek direct evidence that CD4+ CD8alpha alpha + thymocytes represent the normal progeny of human CD4+ CD8- thymocyte precursors in the pathway of T-cell differentiation, highly purified CD3- CD4+ CD8- thymocytes (>98% pure) were analyzed for their developmental fate in the hu/mo FTOC system. The pattern of differentiation obtained in different experiments was identical (Fig 4A): CD4+ cells acquired rapidly the CD8alpha chain, while remaining CD8beta negative (30% CD8alpha +beta -, <2% CD8alpha beta + by day 4) and, later on, CD8beta chain was coexpressed with CD8alpha in a significant cell fraction (40% by day 11; Fig 4A) that became the major population (>90% CD8alpha beta +) by day 15 to 16 (data not shown). More importantly, three-color flow cytometry of the cells harvested on days 4 and 11 of culture extended our findings ex vivo, and confirmed that acquisition of the CD8alpha beta + phenotype was linked to the expression of TCRbeta ic, so that all CD8alpha alpha + progeny generated in the thymic lobes remained negative for TCRbeta ic expression (Fig 4B). As observed when thymic lobes were reconstituted with CD4+ CD8alpha alpha + cells, cellular proliferation lasted for 6 to 7 days in lobes injected with CD3- CD4+ CD8- progenitors. Regardless of the starting population, cellular expansion in the FTOC involved preferentially (if not exclusively) the TCRbeta ic+ CD8alpha beta + population, so that generation of CD8alpha alpha + cells may ocurr essentially in the absence of cell proliferation. Taken together, our results provide formal proof that CD4+ CD8alpha alpha + thymocytes are the direct progeny of CD3- CD4+ CD8- thymocytes and the immediate precursors of the first DP thymocytes expressing the CD8alpha beta heterodimeric form. In addition, they are compatible with a model in which the CD8alpha chain is acquired by developing cells in the human thymus before the process of beta -selection, whereas expression of CD8beta is induced as a developmental consequence of the beta -selection process after signaling through the pre-TCR.


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Fig 4. CD4+ CD8alpha alpha + human thymocytes are functional intermediates between CD3- CD4+ CD8- progenitors and CD4+ CD8alpha beta + beta -selected pre-T cells. (A) CD3- CD4+ CD8- thymocytes, isolated as described in Materials and Methods, were cultured in a hybrid hu/mo FTOC and analyzed after 4 and 11 days for the coexpression of CD4, CD8alpha , and CD8beta . (B) Intracytoplasmic TCRbeta (TCRbeta ic) expression (shaded areas) was analyzed by three-color flow cytometry in the cellular progeny recovered at day 4 of culture. By day 11, analysis was performed after electronic gating on the CD8alpha +beta - and the CD8alpha +beta + progeny. Background fluorescence was determined with isotype-matched irrelevant MoAb. A representative experiment out of five is shown.

Developmental events associated with beta -selection parallel the onset of CD8beta chain expression.   The above data allowed us to propose that CD8beta expression is a cell marker of beta -selection. Further support for this notion came from molecular studies aimed at analyzing individual CD8alpha alpha + and CD8alpha beta + pre-T-cell stages for the expression of several proteins known to be regulated as a consequence of the beta -selection process. Particularly, phosphorylation of Retinoblastoma (Rb), which is tightly associated with cell-cycle activation,30 expression of cell-cycle-associated cyclin A and cyclin B, and downregulation of RAG2 protein expression were analyzed by Western blotting, as indicators of beta -selection (Fig 5 and Table 1).8 As shown in Fig 5, a mixture of both the hyperphosphorylated and the hypohosphorylated Rb forms was detected in CD3- CD4+ CD8- as well as in CD4+ CD8alpha alpha + thymocytes. This pattern changed dramatically at the next CD3low pTalpha + DP stage that showed an exclusive expression of the slow hyperphosphorylated Rb form, which was also the predominant form displayed by large CD3- CD4+ CD8alpha beta + pre-T cells. Expectedly, noncycling, small CD3- DP pre-T cells10 displayed exclusively the fast hypophosphorylated Rb form. Of note, expression of the 60-kD cyclin A, which was barely detectable in CD3- CD4+ CD8- and CD4+ CD8alpha alpha + thymocytes, was markedly upregulated in both the CD3low pTalpha + and the CD3- large DP pre-T-cell subsets displaying an exclusive hyperphosphorylated Rb form, whereas it dropped to basal levels in the CD3- small DP cells. An identical expression pattern was observed for the 62-kD cyclin B (Fig 5).


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Fig 5. Analysis of Rb phosphorylation, and expression of Cyclins A and B and RAG proteins in distinct subsets of human pre-T cells. Cellular lysates from human thymocytes of the indicated phenotypes were isolated as described in Materials and Methods and analyzed by Western blotting for the expression of Rb, Cyclin A, Cyclin B, RAG1, and RAG2. Expression of beta -tubulin was analyzed as an internal control. Molecular sizes are indicated on the left (kD).


                              
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Table 1. Summary of the Developmental Features of Pre- and Post-beta -Selected Human Pre-T Cells

Analysis of RAG2 protein content in individual pre-T-cell subsets indicated that, as reported in mice,8 RAG2 downregulation may be a functional developmental consequence of beta -selection also in humans. As shown in Fig 5, we found that the relative expression levels of RAG2 (as compared with those of beta -tubulin) was reduced in both subsets (CD3low and CD3-) of CD8alpha beta + pre-T cells, and became almost undetectable in downstream small pre-T cells. However, RAG2 protein was reexpressed at high levels at the next stage of CD3-TCRalpha beta -expressing DP thymocytes (data not shown) indicating that, similarly to mice, RAG2 downregulation is transient in thymocyte development in humans. Parallel analysis showed that RAG1 was not subjected to a similar developmental regulation at the protein level, because relatively high levels of RAG1 were expressed in all pre-T-cell subsets. Taken together, these results provide additional support to our proposal that beta -selection occurs in humans during the transition from CD4+ CD8alpha alpha + to CD4+ CD8alpha beta + DP pre-T cells and allow us to conclude that CD8beta chain induction may be one of the direct consequences of the beta -selection process.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The progression of thymocytes through distinct developmental stages is marked by the ordered pattern of expression of a number of cell surface molecules of which the coreceptors CD4 and CD8 are particularly relevant. In humans, the developmental expression of these two molecules marks progression of thymocytes from immature CD4- CD8- DN cells through CD4+ CD8- intermediates and finally to CD4+ CD8+ DP thymocytes, before expression of a surface alpha beta TCR and commitment to either CD4+ CD8- or CD4- CD8+ mature T cells.3,16 We have previously shown that essentially all CD4+ CD8- intermediates display a germline configuration at the TCRbeta locus and lack expression of TCRbeta protein, whereas both TCRbeta gene rearrangements and cytoplasmic TCRbeta chain expression occur in the vast majority of thymocytes at the downstream CD4+ CD8+ TCRalpha beta - stage. The finding that the latter subset is highly enriched in large cycling cells25 allowed us to propose that the CD4+ CD8- to CD4+ CD8+ transition represents the critical developmental point at which beta -selection promotes the clonal expansion and further differentiation of human pre-T cells independent of TCRalpha .25 Molecular support to that notion came from additional studies showing that expression of surface CD3 and pTalpha chain is restricted in vivo to a significant proportion of cells within the population of CD4+ CD8+ TCRalpha beta - cycling thymocytes,10 indicating that the human pre-TCR is actually expressed on pre-T cells in transit to the CD4+ CD8+ DP stage.25 However, we show in this study that the DP compartment is heterogeneous regarding the expression of CD8, so that it is mostly composed of cells with the expected CD4+ CD8alpha beta + phenotype, but also includes a minor fraction of cells that coexpress CD4 and CD8alpha without CD8beta . The physiological existence of such CD4+ CD8alpha +beta - thymocytes suggests that expression of the CD8 glycoprotein either as an alpha alpha homodimer or as an alpha beta heterodimer26 is developmentally controlled and, therefore, the relevance of the CD4+ CD8- to CD4+ CD8+ transition as the critical check-point of beta -selection needs to be revisited.

Confirming the observation by Spits et al,17 we show here that CD4+ CD8alpha alpha + cells are very rare in the human thymus (<2% of total thymocytes), this probably precluding previous studies on their physiological role in thymic T-cell development. We have now approached this issue by taking advantage of the fact that such cells can be enriched (up to 35%) within the fraction of large-sized CD3- DP thymocytes recovered from Percoll density gradients (Fig 1). An important aspect of our study was the finding that no TCRbeta chain was expressed in the cytoplasm of isolated CD4+ CD8alpha alpha + thymocytes, although they showed extensive TCRbeta gene rearrangements (Blom et al,24 and our unpublished results); whereas downstream CD4+ CD8alpha beta + cells were homogeneously TCRbeta ic+. Because TCRbeta ic expression is currently envisaged as a marker of beta -selection, one might conclude that upregulation of CD8beta parallels beta -selection in the human thymus, whereas induction of CD8alpha is not coupled to that critical process. However, a very recent report by Blom et al24 has shown that TCRbeta ic chain is already expressed in a significant proportion (up to 25%) of CD4+ CD8alpha alpha + thymocytes that express very low surface CD3, and may thus be cells that have completed beta -selection. Although it can not be ruled out that TCRbeta ic+ cells characterized by Blom et al24 are actually conventional DP thymocytes that downregulate CD8beta from the cell surface due to the ex vivo isolation procedure, it is also possible that such cells are very recent beta -selected cells upregulating CD8beta in vivo. Alternatively, CD4+ CD8alpha alpha + TCRbeta ic+ cells could have escaped detection in our study. However, we consider this last possibility very unlikely because we consistently observed lack of both TCRbeta ic and surface CD3 not only in CD4+ CD8alpha alpha + cells isolated ex vivo, but also in those generated in vitro in FTOC from the CD4+ CD8- precursor subset. Moreover, we showed that the coincident developmental expression of TCRbeta ic and CD8beta was associated with progression through DNA synthesis and expression of cell-cycle-associated proteins both in vivo (Figs 1 and 5) and in vitro (data not shown), supporting the proposal that CD8beta expression is induced simultaneously to or immediately after beta -selection. A summary of the developmental events associated with progression from CD4+ CD8alpha alpha + TCRbeta ic- cells to CD4+ CD8alpha beta + TCRbeta ic+ cells in humans is illustrated in Table 1.

A second aspect of our study was the functional demonstration of a precursor-product relationship between CD4+ CD8alpha alpha + (TCRbeta ic-) and CD4+ CD8alpha beta + (TCRbeta ic+) thymocytes. This is particularly relevant because CD4+ CD8alpha alpha + thymocytes lacking TCRbeta ic have previously been proposed to be dead-end cells.24 Although our results do not preclude that cell death may physiologically occur within the CD4+ CD8alpha alpha + population in those cells that fail beta -selection because of nonproductive TCRbeta gene rearrangements, they provide direct evidence that CD4+ CD8alpha alpha + TCRbeta ic-_thymocytes represent the immediate precursors of CD4+ CD8alpha beta + TCRbeta ic+ cells and the direct progeny of CD4+ CD8- intermediates. This supports again that CD8beta expression is not induced before the cells have undergone a productive TCRbeta gene rearrangement and express a functional TCRbeta chain. Taken together, the above data allowed us to conclude that beta -selection operates at the CD4+ CD8alpha alpha + to CD4+ CD8alpha beta + transition and, thus, upregulation of CD8beta may be considered as a marker of beta -selection in human thymocyte development. Our proposal has important implications regarding the existence of regulatory mechanisms that account for a differential expression of CD8alpha and CD8beta at distinct stages of thymocyte development. In this regard, it is known that surface expression of CD8beta chain is dependent on CD8alpha chain expression,26 so that both molecules are coordinately expressed on the vast majority of thymocytes and thymus-derived T cells.18,19 However, mechanisms might exist that allow for their discoordinate regulation as well, because other CD8+ cell types such as extrathymically derived intestinal intraepithelial lymphocytes (IEL), and a subset of NK cells express exclusively the CD8alpha alpha homodimeric form.18,31,32 Evidence has been provided that CD8 lineage-specific regulatory sequences direct developmentally correct expression of the human CD8beta gene on thymus-derived T cells from transgenic animals33; and recent data by Littman et al34 have shown that multiple developmental stage-specific enhancers regulate CD8 expression in developing thymocytes and in thymus-independent T cells in mice. These studies, however, have not addressed the question as to how lineage-specificity of CD8alpha and CD8beta expression is achieved. As previously suggested in mice,15 it is likely that regulatory signals provided by the thymic microenvironment are involved in controlling CD8beta expression also in humans. In both species, CD8alpha alpha + thymocytes remain negative for CD8beta in cytokine-supplemented cultures in vitro,15,17 but acquire CD8beta when transferred into a FTOC (Hori et al17 and present study). Interestingly, we show here that progression to the CD8beta + stage is associated with acquisition of low-surface CD3 and, more importantly, of stoichiometric levels of surface pTalpha chain. It is thus tempting to speculate that CD8beta expression in humans is coupled to signalling through the pre-TCR, which might be induced at the CD4+ CD8alpha alpha + to CD4+ CD8alpha beta + transition, to be rapidly downregulated from the cell surface once the cell has been brought into cycle and has attained the DP stage.25 Therefore, as reported for its murine counterpart,7-9 the human pre-TCR may participate in the transition to the conventional CD4+ CD8+ stage, at which expression of the CD8alpha beta heterodimer will be critically involved in the final maturation of MHC class-I-restricted CD8-lineage T cells.19-22


    ACKNOWLEDGMENT

We thank Drs M. Brenner, R. Kurrle, and E.L. Reinherz for the generous gift of antibodies, Dr K. Schwarz for helpful discussions, Dr J.C. Segovia for assistance with cell sorting, and the Pediatric Cardiosurgery Units from the Centro Especial Ramón y Cajal and Ciudad Sanitaria La Paz (Madrid, Spain) for the thymus samples. We also want to express our gratitude to the people of the mouse facilities at our Institute for his continuous support.


    FOOTNOTES

Submitted May 17, 1999; accepted July 16, 1999.

Supported in part by grants from Glaxo Wellcome S.A.; SAF97-0161 from Comisión Interministerial de Ciencia y Tecnología (CICYT); PB97-1194 from Dirección General de Enseñanza Superior e Investigación Científica (DGES); and 08.3/0013/1997 from Comunidad Autónoma de Madrid (CAM). The Centro de Biología Molecular "Severo Ochoa" is partially supported by the Fundación Ramón Areces. Y.R.C., A.R.R., and V.G.Y. are fellows from the Fundación Ramón Areces, Ministerio de Educación y Ciencia, and Fondo de Investigación Sanitaria, respectively.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Address reprint requests to María L. Toribio, PhD, Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain; e-mail: mtoribio{at}cbm.uam.es.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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© 1999 by The American Society of Hematology.
 
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