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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Carrasco, Y. R. Articles by Toribio, M. L. Search for Related Content PubMed PubMed Citation Articles by Carrasco, Y. R. Articles by Toribio, M. L. Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 94 No. 10 (November 15), 1999: pp. 3491-3498 -Selection Is Associated With the Onset of CD8 Chain Expression on CD4+CD8+ 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)  locus are selected for proliferation and further maturation, before TCR expression, by signaling through a pre-TCR composed of the TCR chain paired with a pre-TCR (pT) chain. Such a critical developmental checkpoint, known as -selection, results in progression from CD4 CD8 double negative (DN) to CD4+ CD8+ double positive (DP) TCR 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 and  chains of CD8 at distinct maturation stages. Our results indicate that CD8, but not CD8, is expressed in vivo in a minor subset of DP TCR thymocytes, referred to as CD4+ CD8+ pre-T cells, mostly composed of resting cells lacking cytoplasmic TCR chain (TCRic). In contrast, expression of CD8 heterodimers was selectively found on DP TCR thymocytes that express TCRic and are enriched for cycling cells. Interestingly, CD4+ CD8+ pre-T cells are shown to be functional intermediates between CD4+ CD8 TCRic and CD4+ CD8+ TCRic+ thymocytes. More importantly, evidence is provided that onset of CD8 and TCRic expression are coincident developmental events associated with acquisition of CD3 and pT chain on the cell surface. Therefore, we propose that the CD4+ CD8+ to CD4+ CD8+ transition marks the key control point of pre-TCR-mediated -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  and  T-cell receptor (TCR) genes to finally generate mature T lymphocytes bearing an TCR.1-3 Early during the intrathymic developmental process, pre-T cells that have succeeded in productive rearrangements at the TCR locus are rescued from apoptotic cell death and selected for further maturation, before TCR expression, by signaling through a pre-TCR composed of the TCR chain paired with a pre-TCR (pT) 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 TCR chain, a process that has been termed -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 TCR 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 CD8 chain in response to certain combinations of cytokines,14 thus expressing the CD8 homodimeric form. Interestingly, such CD8+ cells remain negative for CD3 and CD4 in vitro, but can differentiate into conventional CD4+ CD8+ DP thymocytes expressing the CD3-associated 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 CD8 homodimer17 but generate common CD4+ CD8+ DP thymocytes with the mature CD3-TCR complex in vivo.16 Current data on the physiologic expression of CD8 either as an homodimer or as an heterodimer on distinct CD8+ cell types support the notion that CD8+ T cells, such as intestinal intraepithelial lymphocytes (IEL), can be generated by an extrathymic maturation pathway independent of CD8 expression, whereas induction of CD8 and, hence, the generation of CD8+ cells is thymus-dependent.18,19 This is further supported by functional studies showing that the CD8 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 CD8/ mice.19-22 All these data concur with the fact that the CD8+ 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 CD8+.15,19,23 Nonetheless, CD8+ 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 CD8+ thymocytes, which coexpress surface CD4 but still lack a mature TCR,7 have extensive TCR gene rearrangements. However, functional studies on the developmental potential and precursor-product relationships of isolated CD4+ CD8+ thymocytes are still lacking, precluding a better understanding of the physiological relevance of this particular cell subset in the context of thymocyte development and -selection in humans. Here we show that CD4+ CD8+ human thymocytes are actually functional intermediates between CD4+ CD8 and CD4+ CD8+ thymocytes. More importantly, evidence is provided that onset of CD8 expression and -selection are coincident events associated with the acquisition of CD3 and pT chain on the cell surface during intrathymic development in humans. These results suggest that the pre-TCR-mediated check-point of -selection is placed in humans at the CD4+ CD8+ to CD4+ CD8+ 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 TCR and CD3 expression, and will be hereafter referred to as small DP CD3 thymocytes.10 In contrast, large DP thymocytes included CD3 and CD3low pT+ thymocytes. Both subsets were independently isolated from the whole pool of large DP TCR 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 CD8+ and CD8+ 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), CD8 (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 CD8 (2ST8-5H7, kindly provided by Dr E.L. Reinherz, Dana-Farber Cancer Institute, Boston, MA)26 as well as MoAb recognizing monomorphic determinants of TCR (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 TCR, cells were treated with 0.5% saponin (Sigma, St Louis, MO), incubated with the anti-TCR chain 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 pT 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 CD8+ and CD8+ cells was performed in an EPICS Elite Cell Sorter (Coulter Electronics, Inc) on isolated large DP CD3 thymocytes after labeling with anti-CD8 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--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 TCR+ 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 CD8+ 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+ CD8+ DP cells in the human thymus.   We have recently reported that surface CD3 expression and cell size define three subsets of human TCR 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 CD8 molecule), so that they were characterized as DP thymocytes.10 However, as shown in Fig 1A, analysis on the correlated expression of CD8 and CD8, performed with the anti-CD8 MoAb 2ST8-5H7,26 showed that large CD3low and small CD3 pre-T cells were homogeneously CD8++, whereas a significant proportion (32 ± 4%) of large CD3 pre-T cells expressed CD8 but lacked CD8 chain. Such a differential expression of the  and  chains of CD8 defines two distinct subsets of large CD3 pre-T cells in which CD8 may be expressed either as a CD8 homodimer or as a CD8 heterodimer,26 so that they will hereafter be referred to as either CD8+ or CD8+, 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 TCR chain (TCRic). As shown in Fig 1B, a clear phenotypic pattern was obtained: TCRic expression was not detectable in CD8+ thymocytes, whereas essentially all CD8+ pre-T cells coexpressed TCRic. These results confirm our previous data showing a bimodal distribution of TCRic within the pool of large CD3 pre-T cells as a whole10 but, in addition, they suggest that expression of TCRic parallels acquisition of the CD8+ phenotype. View larger version (30K): [in this window] [in a new window]   Fig 1. Differential expression of CD8 and CD8 chains on distinct subsets of human pre-T cells: Intracytoplasmic TCR expression and cell-cycle progression are associated with CD8 chain expression. (A) Large CD3, large CD3low pT+, and small CD3 pre-T cells, isolated as described in Materials and Methods, were analyzed by two-color flow cytometry for CD8 versus CD8 expression. (B) Large CD3 CD4+ CD8+ (DP) pre-T cells were fractionated by cell sorting into CD8+(top panels) and CD8++ (bottom panels) cells after labeling with the 2ST8-5H7 anti-CD8 MoAb plus PE-coupled goat antimouse IgG2a. Reanalysis of surface CD8 expression postsorting is shown (shaded histograms). Sorted cells were then analyzed by flow cytometry for intracytoplasmic TCR chain (TCRic) 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 TCRic expression is currently envisaged as a marker of -selection, we concluded that the CD8+ and the CD8+ subsets could represent two distinct pre-T-cell developmental stages placed on either side of the -selection process. This was confirmed by FCA showing that both populations differed dramatically in their cell cycle status. As expected of -selected thymocytes, CD8+ pre-T cells featured a high proportion (up to 55%) of cycling cells, whereas essentially all (>90%) CD8+ thymocytes were "unselected" cells arrested in the G0/G1 phases of the cell cycle (Fig 1B). The distinct developmental status of the CD8+ and CD8+ 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 -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 -selected CD8+ TCRic+ thymocytes were homogeneously positive for the expression of CD44, whereas CD8+ 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 CD8+ pre-T cells, whereas CD5 and CD69 were similarly expressed in the three populations (Fig 2). View larger version (28K): [in this window] [in a new window]   Fig 2. Cell-surface phenotype of individual subsets of CD8+and CD8+ 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 CD8+ (shaded areas) or CD8++ (unshaded areas, bold line) cells. Large CD3low pT+ pre-T cells, shown to be homogeneously CD8++ (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+ CD8+ pre-T cells are the immediate precursors of CD4+ CD8+ pre-T cells that coexpress surface pT and CD3.   To provide direct evidence that CD4+ CD8+ 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+ CD8+ thymocytes (>98% pure after cell sorting) were consistently found to give rise to conventional DP cells coexpressing CD4 and the CD8 heterodimer at early periods of culture (20% by day 5 in this experiment; Fig 3A, upper panel). Of notice, essentially all CD4+ CD8+ progeny (>95%) generated by this time in different experiments had acquired low surface CD3 with minimal differentiation (<2%) into TCR+ cells (Fig 3A, lower panel). Interestingly, such CD3low TCR CD4+ CD8+ pre-T cells (20% of total cells recovered by day 5) were all positive for the expression of TCRic and, more importantly, they displayed low surface levels of the pT chain (Fig 3B), as assessed with a polyclonal anti-pT antibody.10 View larger version (33K): [in this window] [in a new window]   Fig 3. Phenotypic analysis of the cellular progeny generated after differentiation of CD4+ CD8+ human pre-T cells in FTOC. (A) CD4+ CD8+ 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 CD8, CD8, TCR, and CD3. Analysis of TCR versus CD3 was performed by three-color flow cytometry after electronic gating on the CD8+ progeny. (B) Intracytoplasmic TCR (TCRic) and surface pT 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+ CD8+ pre-T cells with this particular CD3low pT+ phenotype are functional progenitors of common DP thymocytes that already express surface TCR.25 Accordingly, CD4+ CD8+ 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 TCR (50% of total cells recovered by day 14 in the experiment shown in Fig 3A). These results provide evidence that CD4+ CD8+ thymocytes represent the immediate precursors of the first intrathymic cells with a conventional CD4+ CD8+ DP phenotype. Moreover, they indicate that the developmental onset of CD8 chain expression is closely associated with the -selection process and parallels the acquisition of a surface pre-TCR. CD4+ CD8+ pre-T cells are functional intermediates between CD4+ CD8 progenitors and CD4+ CD8+ pre-T cells.   It is currently believed that CD4+ CD8 thymocytes that still lack the CD3-TCR are the immediate precursors of DP thymocytes in the human thymus.16 However, we show in this study that CD4+ CD8+ pre-T cells are efficient progenitors of CD4+ CD8+ DP thymocytes, suggesting that they represent very transient intermediates between CD4+ CD8 and CD4+ CD8+ cells in vivo. To seek direct evidence that CD4+ CD8+ 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 CD8 chain, while remaining CD8 negative (30% CD8+, <2% CD8+ by day 4) and, later on, CD8 chain was coexpressed with CD8 in a significant cell fraction (40% by day 11; Fig 4A) that became the major population (>90% CD8+) 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 CD8+ phenotype was linked to the expression of TCRic, so that all CD8+ progeny generated in the thymic lobes remained negative for TCRic expression (Fig 4B). As observed when thymic lobes were reconstituted with CD4+ CD8+ 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 TCRic+ CD8+ population, so that generation of CD8+ cells may ocurr essentially in the absence of cell proliferation. Taken together, our results provide formal proof that CD4+ CD8+ thymocytes are the direct progeny of CD3 CD4+ CD8 thymocytes and the immediate precursors of the first DP thymocytes expressing the CD8 heterodimeric form. In addition, they are compatible with a model in which the CD8 chain is acquired by developing cells in the human thymus before the process of -selection, whereas expression of CD8 is induced as a developmental consequence of the -selection process after signaling through the pre-TCR. View larger version (32K): [in this window] [in a new window]   Fig 4. CD4+ CD8+ human thymocytes are functional intermediates between CD3 CD4+ CD8 progenitors and CD4+ CD8+ -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, CD8, and CD8. (B) Intracytoplasmic TCR (TCRic) 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 CD8+ and the CD8++ progeny. Background fluorescence was determined with isotype-matched irrelevant MoAb. A representative experiment out of five is shown. Developmental events associated with -selection parallel the onset of CD8 chain expression.   The above data allowed us to propose that CD8 expression is a cell marker of -selection. Further support for this notion came from molecular studies aimed at analyzing individual CD8+ and CD8+ pre-T-cell stages for the expression of several proteins known to be regulated as a consequence of the -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 -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+ CD8+ thymocytes. This pattern changed dramatically at the next CD3low pT+ DP stage that showed an exclusive expression of the slow hyperphosphorylated Rb form, which was also the predominant form displayed by large CD3 CD4+ CD8+ 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+ CD8+ thymocytes, was markedly upregulated in both the CD3low pT+ 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). View larger version (57K): [in this window] [in a new window]   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 -tubulin was analyzed as an internal control. Molecular sizes are indicated on the left (kD).                                View this table: [in this window] [in a new window]   Table 1. Summary of the Developmental Features of Pre- and Post--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 -selection also in humans. As shown in Fig 5, we found that the relative expression levels of RAG2 (as compared with those of -tubulin) was reduced in both subsets (CD3low and CD3) of CD8+ 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-TCR-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 -selection occurs in humans during the transition from CD4+ CD8+ to CD4+ CD8+ DP pre-T cells and allow us to conclude that CD8 chain induction may be one of the direct consequences of the -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 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 TCR locus and lack expression of TCR protein, whereas both TCR gene rearrangements and cytoplasmic TCR chain expression occur in the vast majority of thymocytes at the downstream CD4+ CD8+ TCR 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 -selection promotes the clonal expansion and further differentiation of human pre-T cells independent of TCR.25 Molecular support to that notion came from additional studies showing that expression of surface CD3 and pT chain is restricted in vivo to a significant proportion of cells within the population of CD4+ CD8+ TCR 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+ CD8+ phenotype, but also includes a minor fraction of cells that coexpress CD4 and CD8 without CD8. The physiological existence of such CD4+ CD8+ thymocytes suggests that expression of the CD8 glycoprotein either as an homodimer or as an heterodimer26 is developmentally controlled and, therefore, the relevance of the CD4+ CD8 to CD4+ CD8+ transition as the critical check-point of -selection needs to be revisited. Confirming the observation by Spits et al,17 we show here that CD4+ CD8+ 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 TCR chain was expressed in the cytoplasm of isolated CD4+ CD8+ thymocytes, although they showed extensive TCR gene rearrangements (Blom et al,24 and our unpublished results); whereas downstream CD4+ CD8+ cells were homogeneously TCRic+. Because TCRic expression is currently envisaged as a marker of -selection, one might conclude that upregulation of CD8 parallels -selection in the human thymus, whereas induction of CD8 is not coupled to that critical process. However, a very recent report by Blom et al24 has shown that TCRic chain is already expressed in a significant proportion (up to 25%) of CD4+ CD8+ thymocytes that express very low surface CD3, and may thus be cells that have completed -selection. Although it can not be ruled out that TCRic+ cells characterized by Blom et al24 are actually conventional DP thymocytes that downregulate CD8 from the cell surface due to the ex vivo isolation procedure, it is also possible that such cells are very recent -selected cells upregulating CD8 in vivo. Alternatively, CD4+ CD8+ TCRic+ cells could have escaped detection in our study. However, we consider this last possibility very unlikely because we consistently observed lack of both TCRic and surface CD3 not only in CD4+ CD8+ 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 TCRic and CD8 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 CD8 expression is induced simultaneously to or immediately after -selection. A summary of the developmental events associated with progression from CD4+ CD8+ TCRic cells to CD4+ CD8+ TCRic+ 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+ CD8+ (TCRic) and CD4+ CD8+ (TCRic+) thymocytes. This is particularly relevant because CD4+ CD8+ thymocytes lacking TCRic 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+ CD8+ population in those cells that fail -selection because of nonproductive TCR gene rearrangements, they provide direct evidence that CD4+ CD8+ TCRic_thymocytes represent the immediate precursors of CD4+ CD8+ TCRic+ cells and the direct progeny of CD4+ CD8 intermediates. This supports again that CD8 expression is not induced before the cells have undergone a productive TCR gene rearrangement and express a functional TCR chain. Taken together, the above data allowed us to conclude that -selection operates at the CD4+ CD8+ to CD4+ CD8+ transition and, thus, upregulation of CD8 may be considered as a marker of -selection in human thymocyte development. Our proposal has important implications regarding the existence of regulatory mechanisms that account for a differential expression of CD8 and CD8 at distinct stages of thymocyte development. In this regard, it is known that surface expression of CD8 chain is dependent on CD8 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 CD8 homodimeric form.18,31,32 Evidence has been provided that CD8 lineage-specific regulatory sequences direct developmentally correct expression of the human CD8 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 CD8 and CD8 expression is achieved. As previously suggested in mice,15 it is likely that regulatory signals provided by the thymic microenvironment are involved in controlling CD8 expression also in humans. In both species, CD8+ thymocytes remain negative for CD8 in cytokine-supplemented cultures in vitro,15,17 but acquire CD8 when transferred into a FTOC (Hori et al17 and present study). Interestingly, we show here that progression to the CD8+ stage is associated with acquisition of low-surface CD3 and, more importantly, of stoichiometric levels of surface pT chain. It is thus tempting to speculate that CD8 expression in humans is coupled to signalling through the pre-TCR, which might be induced at the CD4+ CD8+ to CD4+ CD8+ 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 CD8 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. 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