Expression of CD21 is developmentally regulated during thymic maturation of human T lymphocytes
Elizabeth M. Fischer,
Amal Mouhoub,
Françoise Maillet,
Véronique Frémeaux-Bacchi,
Corinne Krief,
Hannah Gould1,
Sonia Berrih-Aknin2 and
Michel D. Kazatchkine
INSERM U430, Hôpital Broussais, 96 rue Didot, 75014 Paris, France
1 Developmental Biology Research Center, The Randall Institute, King's College, London WC2B 5RL, UK
2 CNRS URA 1159, Hôpital Marie Lannelongue, 92350 Le Plessis Robinson, France
Correspondence to:
E. Fischer
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Abstract
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CD21, the C3d/CD23/EpsteinBarr virus (EBV), receptor is expressed at low density on cells of the T lineage. Immature thymocytes express CD21 with high density. In the present study, we have analyzed the expression of CD21 during intrathymic maturation of T cells. An intense staining for CD21 was observed at the double-negative stage and at the stage of early acquisition of CD4. CD21 expression was decreased at the double-positive and single-positive stages, to then reach levels similar to those of peripheral blood T cells. Staining of thymus sections showed a bright fluorescent signal on thymocytes entering the thymus in the cortical region. Taking advantage of the immature phenotype of cells expressing high amounts of CD21 (CD21++), we depleted thymocyte suspensions in CD3+ and CD8+ cells to study the properties of CD21 on this cell subset. Triggering of CD21 with its ligands iC3b, CD23 and anti-CD21 mAb did not alter the proliferative response of thymocytes to IL-7, and did not induce the differentiation of early cells into CD4+CD8+ thymocytes. Immunoprecipitation did not reveal any molecule associated with CD21 that could play a signaling role in thymocytes. Finally, EBV induced a down-regulation of CD21 and an up-regulation of CD1 in CD21++ thymocytes. Taken together, our observations demonstrate a regulated expression of CD21 on human thymocytes and suggest that the CD21++ subset may be a target for EBV. We further suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.
Keywords: CD23, EpsteinBarr virus, thymocytes, maturation
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Introduction
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The CD21 molecule [the complement receptor type 2 (CR2)] functions as a receptor for the surface-bound iC3b/C3dg/C3d fragments of human C3, for the low-affinity receptor for IgE, CD23, and for EpsteinBarr virus (EBV) (13). CD21 is a transmembrane molecule of 145 kDa, consisting of a large extracellular domain of 15 or 16 tamdemly repeated short consensus repeat sequences followed by a transmembrane region and a short intracytoplasmic domain (4). In the membrane of mature B cells, CD21 is in the form of a stable complex with CD19 and TAPA-1 (5,6). The interaction of CD21 with C3 and CD23 was shown to enhance B cell activation, proliferation, differentiation into IgE-producing cells and to rescue germinal center B cells from apoptosis (2,79). The binding of EBV to CD21 on B cells is followed by endocytosis of the CD21/virus complex (10,11). EBV infection of B cells alters their growth, which may result in autoantibody production and in B cell malignancy (12,13).
In addition to B cells, CD21 is expressed on thymocytes, a subset of peripheral T lymphocytes, follicular dendritic cells, astrocytes and some epithelial cells (1418). The functions of CD21 on human cells of the T lineage remain unclear. CD21 was, however, demonstrated to mediate an increased concentration of Cai2+, and to cap and internalize ligands on cells of the human HPB-ALL T cell line (19). We have previously demonstrated that the expression of CD21 extends to both immature and mature thymocytes, and identified an immature subset of CD3 CD1+ large thymocytes that expresses CD21 with high density, suggesting a role for CD21 in the proliferation and/or differentiation of early thymocytes (20). In the present study, we demonstrate that CD21 is differentially expressed on thymocytes during thymic maturation. We have enriched thymocytes in immature CD21++ cells to investigate the functional consequences of the ligation of the receptor with iC3b, CD23 and EBV.
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Methods
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Antibodies and reagents
Anti-CD21 mAb HB5 (ATCC, Rockville, MD), BL13 (a gift from J. Brochier, Montpellier, France) and anti-CD35 mAb J3D3 (21) were biotinylated using biotin-X-NHS (France Biochem, Meudon, France. The isotypic control mAb MOPC21 (IgG1) and UPC10 (IgG2a) were purshased from Cappel (Cochranville, PA). FITC-conjugated mAb against CD2 (Leu5b), CD3 (Leu4), CD4 (Leu3a), CD34 (HPCA-2), CD19, phycoerythrin (PE)-conjugated mAb against CD8 (Leu2a) and streptavidin(SA)PE were obtained from Becton Dickinson (Le Pont de Claix, France). FITCanti-CD45 mAb and FITCanti-CD1a mAb (OKT6) were purchased from Immunotech (Marseille, France) and Ortho Diagnostics (Raritan, NJ) respectively. Recombinant human IL-7 and IL-1ß were from Genzyme (Cambridge, MA).
Immunofluorescence
For immunofluorescence studies, cells (3x105) were stained with fluorescent or with biotinylated (0.5 µg) mAb in PBS containing 2% normal AB serum for 30 min at 4°C. Biotinylated mAb were revealed by further incubation with SAPE for 20 min. After two washes, cells were fixed in PBS containing 1% formaldehyde. Fluorescence was analyzed using a FACScan (Becton Dickinson) and the CellQuest software. Statistical analysis was performed using Student's t-test.
In situ, fluorescent studies were performed on frozen sections. Acetone-fixed 5 mm frozen thymic sections were incubated with biotinylated anti-CD21 mAb HB5 (dilution 1/3) overnight, followed by three washes in PBS and then revealed with avidinFITC (Amersham, Les Ulis, France) or avidinTexas Red (Amersham) at a 1/100 dilution. Three antibodies were used for double-staining analysis: (i) to visualize the epithelial network, polyclonal anti-keratin antibodies (Dako, Trappes, France) were used at a 1/100 dilution and revealed with goat anti-mouse IgTRITC (Immunotech) at a 1/60 dilution for 30 min; (ii) to visualize the vessel structures, mouse monoclonal anti-
-smooth muscle actin antibodies (Dako) were used at a 1/50 dilution and then revealed with rat anti-mouse IgTexas Red (Dako) at 1/40 dilution for 30 min; and (iii) to visualize the thymic precursor cells, anti-CD34 mAb coupled to FITC (Becton Dickinson) was used at half dilution for 60 min. Sections were then washed 3 times in PBS and mounted under coverslips with glycerol/PBS. Control sections were incubated with the fluorescent conjugates. We also checked that same the results were obtained in simple- and double-fluorescence experiments.
Cell isolation
Human thymus (discarded tissue) was obtained from children <1 year of age undergoing cardiac surgery. Thymus tissue was minced and cells were collected by Ficoll-Hypaque gradient centrifugation. For phenotypic analysis, thymocytes were positively selected by rosetting with AET-treated sheep red blood cells. For isolation of thymocytes expressing CD21 at high density, the thymocyte suspension was sequentially incubated in RPMI containing 1% SVF with magnetic beads (Dynabeads M-450; Dynal, Compiègne, France) coated with anti-CD3 (3 beads/cell), anti-CD8 (4 beads/cell) and anti-CD19 (1 bead/cell) for 30 min at 4°C.
Culture conditions
The iC3b fragment of human C3 was obtained as previously described (22). The 25 kDa recombinant form of soluble (s) CD23 was a gift of J. Y. Bonnefoy (Glaxo, Plan les Ouates, Switzerland). Murine CD23- and mock-transfected L cells were cultured under selection pressure as previously reported (23). Thymocytes that had been depleted in CD3+, CD8+ and CD19+ cells were cultured at 0.5x106/ml in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% FCS in flat-bottom 96-well microplates. In selected experiments, thymocytes were incubated with rIL-7 (0.55ng/ml) in the presence of iC3b (20 µg/ml) or sCD23 (10 µg/ml). In additional experiments, iC3b and sCD23 were coated on the culture microplate for 18 h at 4°C prior to addition of the cells. Thymocytes were also co-cultured at different cell to cell ratios with CD23- or mock-transfected L cells that had been pretreated for 2 h with 50 µg/ml mitomycin C, in the presence of IL-7 or IL-1ß (100 U/ml). Cultures were maintained for 3 days in 5% CO2 at 37°C before being pulsed with 0.5 µCi of [3H]thymidine (ICN, Orsay, France) for 16 h. Incorporation of thymidine was measured in a Micro-ß plate (Wallac, Turku, Finland).
Immunoprecipitation
Thymocytes expressing CD21 at high density (CD21++) (107/ml) were reacted with PBS containing biotin-X-LHS 5 mM for 30 min at 4°C and washed twice with PBS containing 20 mM glycine. The cells were then lysed with 20 mM Tris, 150 mM NaCl, 1 mM EDTA buffer, pH 7.5, containing 0.1% Brij 96, and 1 mM PMSF, 2 µM leupeptin, 10 µM pepstatin and 8 µg/ml aprotin. The lysate was submitted to four preclearing steps using SepharoseProtein coated with control mAb UPC10 and MOPC21 (5 µg of each). Immunoprecipitation was then performed either with anti-CD21 mAb HB5 and BL13 (5 µg of each) or with isotype-matched mAb for 2 h at 4°C. Immunoprecipitates were electrophoresed on 8% SDSPAGE under reducing conditions and then blotted on nitrocellulose membranes (Schleicher, Dassel, Germany) in 25 mM Tris/192 mM glycine buffer containing 20% ethanol for 90 min. The membranes were saturated, incubated with SA peroxidase (Amersham) and then revealed by chemiluminescence using the ECL kit (Amersham). Ten million Raji B lymphoblastoid cells (ATCC) were processed in a parallel fashion as a positive control.
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Results
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Stage-specific expression of CD21 during human thymocyte differentiation
We first confirmed our previous observations of the presence of two distinct subpopulations of human thymocytes, one expressing CD21 with high (CD21++) intensity and the other expressing low amounts of CD21 (CD21+), by analyzing CD2+ cells isolated from 11 freshly obtained human thymuses (20). High levels of CD21 (MFI: 320 ± 42) were expressed by 11 ± 5% thymocytes, whereas 58 ± 10% of cells expressed a low density of the receptor (MFI: 48 ± 6). In order to analyze the expression of CD21 during T cell differentiation, we then used three-color staining with FITCLeu3a, PerCPLeu2a and biotinylated HB5 mAb. We defined discrete stages of differentiation based on the expression of CD4 and CD8, and analyzed the relative intensity of staining for CD21 antigen on gated cells. Figure 1
shows a representative staining experiment performed on CD2+ thymocytes. The findings obtained upon analysis of cells of nine thymuses are summarized in Fig. 2
. The percentage of cells expressing CD21 did not differ significantly between CD4CD8 double-negative (DN) cells (67 ± 6%), intermediate CD4lowCD8 cells (62 ± 7%) and CD4+CD8+ double-positive (DP) common thymocytes (65 ± 6%). The proportion of CD21-expressing cells was, however, markedly decreased upon differentiation of DP cells into CD4+ single-positive (SP) cells (34 ± 5%), although it remained stable upon differentiation of DP cells into CD8+ SP cells (75 ± 5%). The mean fluorescent intensity (MFI) of staining for CD21 was significantly higher on CD4CD8 DN cells (305 ± 79) and CD4lowCD8 cells (219 ± 66) than on cells at later stages of differentiation (Fig. 2
). The density of CD21 on thymocytes was similar on DP CD4+CD8+ cells (98 ± 28), SP CD4+ cells (88 ± 25) and SP CD8+ cells (73 ± 21). There was increased expression of CD21 on CD4+CD8low cells (134 ± 21), which may reflect the heterogeneity of this population that contains cells reaching the DP stage and cells losing the expression of the CD8 marker to become SP CD4+ cells. Staining with anti-CD45 antibodies indicated that CD45RA+ thymocytes expressed CD21 with a higher intensity (230 ± 100) than CD45RO+ cells (87 ± 25).

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Fig. 1. Expression of the CD21 antigen on thymocytes during thymic differentiation. Thymocytes were positively selected for expression of the CD2 antigen prior to analysis by three-color immunofluorescence using FITCanti-CD4 mAb Leu3a, PerCPanti-CD8 mAb Leu2a and biotinylated anti-CD21 mAb HB5. Cells were first gated based on the expression of CD4 and CD8 (upper panel), and further analyzed for the expression of CD21 (lower panels). Dotted histogram (R2, CD4CD8 panel) represents fluorescence (FL2) of cells that had been stained with biotinylated isotypic control mAb UPC10.
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Fig. 2. Expression of CD21 on thymocytes during differentiation. CD2+ thymocytes of nine freshly obtained thymuses were stained as described in Fig. 1 . The MFI of staining is plotted at each stage of differentiation, as indicated on the abcissa. The MFI was significantly higher on CD4CD8 as compared with cells at other stages of differentiation (P < 0.0001) and on CD4lowCD8 as compared with cells at other stages of differentiation (P ranging between 0.0001 and 0.005).
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Staining of total thymic cell suspensions for CD21 revealed that, in addition to T cells, 1.5% of CD21+ cells were B cells co-expressing CD19 and CD21 (MFI for CD21: 537). No staining for CD21 was observed on CD19CD2 cells, indicating that thymocyte precursors and stromal cells in the thymus do not express the CD21 receptor (data not shown). Taken together, our data indicate that the expression of CD21 is regulated throughout thymocyte differentiation, with maximal expression at the CD4CD8 DN stage and at the early stage of acquisition of the CD4 marker by thymocytes.
In order to localize CD21+ cells, we performed double-staining experiments on frozen thymic sections. Double staining with anti-
-smooth muscle actin antibodies indicated that CD21+ cells are located around the vessels (Fig. 3
). Anti-CD21 antibody not only stains some thymocytes but also recognizes some structure proteins on the vessel (Fig. 3
). Anti-keratin antibodies were used to identify the epithelial network. CD21+ cells were found to be close to the subcapsular epithelial network, particularly at sites where the epithelial subcapsular line presents discontinuities (Fig. 3
). Finally, double staining with anti-CD34 antibodies, identifying early thymic precursor cells, demonstrated that some CD21+ cells in the thymus, co-express CD34. The localization of CD21+ cells close to the vessels and discontinuities of the epithelial subcapsular line are compatible with the hypothesis that the CD21+ cells have recently migrated.

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Fig. 3. Expression of CD21 in frozen sections of human thymus. (a and b) Sections were double stained with antibodies to -smooth muscle actin (red) and anti-CD21 antibodies (green). (c and d) Staining for keratin (red) and for CD21 (green); (e and f) staining for CD34 (green) and for CD21 (red); (g and h) negative control using the revealing antibodies alone. A high magnification of the area indicated by a square is shown in (b), (d), (f) and (h). The bars represent 50 µm.
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Properties of thymocytes selected for the expression of CD21 at high density
Thymocytes were enriched in CD21++ cells by depleting thymic cell suspensions in CD3+, CD8+ and CD19+ cells, using magnetic beads coated with the relevant mAb. The selection process was performed on five thymuses, yielding ~5% of the initial thymocyte number. The phenotype of the CD21++-enriched cells obtained in this way is depicted in Fig. 4
. CD21++ cells expressing a MFI of 288, accounted for 71% of the cells. CD21++-enriched cells positively stained for the CD2 antigen, did not express CD3 nor CD8, and did not contain DP CD4+CD8+ thymocytes. Forty-four percent of the CD21++-enriched cells expressed CD4, 23% expressed CD34, 9% expressed CD69, 18% expressed CD45RA and 17% expressed CD45RO. CD21++-enriched cells displayed a larger size (FSC: 511) than unfractionated thymocytes (FSC: 386) (data not shown).

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Fig. 4. Phenotypic characterization of the subpopulation of thymocytes enriched in CD21++ cells. A freshly obtained thymocyte suspension was subjected to negative selection using magnetic beads coated with anti-CD3, anti-CD8 and anti-CD19 mAb. The phenotype of the resulting population was then assessed using two-color fluorescence for the expression of CD2, CD3, CD4, CD8 and CD21.
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The CD21++ thymocytes were then used in functional experiments. The cells were found not to proliferate in the presence of IL-1, IL-2 or IL-4, nor in the presence of anti-CD2 mAb (data not shown). CD21++ thymocytes responded, however, to IL-7, a key growth-promoting factor for the expansion of immature CD3TCR thymocytes (24) (Fig. 5
). Proliferation was decreased upon depletion of the cell suspension in CD34+ cells. We further investigated whether the ligation of CD21 on CD21++ thymocytes modulates their ability to respond to IL-7. No effect of triggering CD21 with anti-CD21 mAb HB5 and BL-13, recombinant CD23 or purified iC3b, whether added in soluble form or immobilized on culture wells, was observed with regard to IL-7-induced proliferation (data not shown). In addition, the co-culture of thymocytes with CD23-transfected murine L cells did not alter the rate of IL-7-induced proliferation of CD21++ thymocytes as compared with that of cultures performed in the presence of wild-type L cells (data not shown).
Since CD23 has been reported to induce the differentiation of pro-thymocytes in the presence of IL-1 (25), we cultured CD21++ thymocytes with CD23- or mock-transfected murine L cells in the presence of recombinant human IL-1ß (100 U/ml), and assessed the cells for the expression of CD3, CD4, CD8, CD1, CD69, CD38 and CD34 after 3 days of culture. No change in phenotype of the cells was observed (data not shown).
We then assessed the capacity of EBV, as an additional CD21 stimulus, to modulate the phenotype of CD21++ thymocytes. EBV induced a dramatic decrease in the expression of CD21 (Fig. 6
). A selective decrease in cells expressing CD21 at high density was also observed in CD3+-depleted thymocytes, suggesting that CD21++ thymocytes are the preferential target for virus binding and internalization. The expression of CD1 and, to a lesser extent, of CD4 and CD8, was increased. Of note, however, was the fact that a part of thymocytes that were CD8 and CD4low at the initiation of the culture acquired these markers upon 3 days of culture in the absence of EBV. The expression of CD3 was unchanged, whether the cells had been cultured in the presence or absence of EBV (data not shown). Although no proliferation of cells was induced by EBV, we observed a 2-fold increase in the viability of CD21++ thymocytes cultured in the presence of EBV for 3 days, as compared with cells cultured in medium alone.

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Fig. 6. Phenotypic changes induced by EBV infection of CD21++ thymocytes. CD21++ thymocytes were incubated for 72 h in the absence (dark histograms) or presence (white histograms) of EBV-containing B95-8 supernatants. The cells were then stained with FITCLeu4a (CD4), FITCLeu2a(CD8), FITCOKT6 (CD1) mAb, and biotinylated HB5 (CD21), J3D3 (CD35) and UPC10 (control) mAb followed by SAPE.
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Since CD21 signals in B cells through a complex involving CD19 and TAPA-1, we investigated whether CD21 is associated with a transducing protein in the membrane of thymocytes. Membranes of CD21++ thymocytes were lyzed under mild conditions using Brij 96. As shown in Fig. 7
, anti-CD21 mAb immunoprecipitated a single protein of 145 kDa from the lysates, corresponding to CD21. Under the same experimental conditions, anti-CD21 mAb immunoprecipitated CD19, TAPA-1, p 40 and p 130 in addition to CD21, in Raji cell lysates, as previously described by Matsumoto et al. (5).

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Fig. 7. Immunoprecipitation of CD21 from CD21++ thymocytes. CD21++ thymocytes were labeled with biotin, lyzed in Tris buffer containing 0.1% Brij 96 before immunoprecipitation with HB5 (d) or isotype-matched (c) mAb. The immunoprecipitates were electrophoresed and blotted. The membrane was revealed with SAPE followed by ECL. Panels (a) and (b) depict the results of the immunoprecipitation of Raji cell lysates.
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Discussion
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In the present study, we demonstrate that, as it is known to be the case for cells of the B lineage, the expression of the CD21 receptor is developmentally regulated during maturation of human T lymphocytes.
In contrast to B cells that maximally express CD21 at a mature stage, thymocytes express the highest density of CD21 at the most immature DN stage of intra-thymic differentiation as well as at the stage of early acquisition of the CD4 antigen. The density of CD21 on DN thymocytes was found to be in the same range as that expressed on the few B cells present in the thymus. It is unlikely that the high expression of the molecule was merely related to the large size of immature thymocytes. Thus, small B cells in early G1 phase express a greater density of receptor per unit membrane than larger G2/M and S phase cells (26); in addition, the density of CD35 was similar on immature and mature thymocytes, irrespective of size (A. Mouhoub, unpublished data). Upon transition to the DP stage the expression of CD21 decreased so as to reach, at further stages of differentiation, a density equivalent to that observed on peripheral blood T lymphocytes (16). However, whereas the percentage of CD21+ cells is similar on peripheral blood CD4+ and CD8+ lymphocytes, we found it to be significantly higher on SP CD8 thymocytes than SP CD4-expressing thymocytes. These results suggest that the expression of CD21 on CD8+ thymocytes is lost at the time of their last phenotypic switch before the cells emigrate from the thymus to the periphery or, alternatively, that some CD8+CD21+ thymocytes become trapped in a secondary lymphoid organ. The immature phenotype of thymocytes that express the highest density of CD21 was further confirmed by their localization in the sub-capsular region of the thymus, close to vessels and by the co-expression of the CD34 antigen by some of the cells. The present observations together with previous data from our laboratory (20) and from Schmitt et al. (27) suggest the following sequence of changes in phenotypic patterns during intra-thymic differentiation of human T lymphocytes: CD34+CD7++CD21++CD1+CD4CD8
CD34CD7++CD21++CD1+CD4lowCD8
CD34CD21+CD1++CD4+CD8+/
CD21+CD1+/CD4+CD8 or CD21+CD1+/CD4CD8 +.
The expression of CD21 is tightly controlled during the maturation process of B lymphocytes. Whereas the receptor is undetectable on immature B cells, it is fully expressed on IgM+ IgD+ mature cells and then lost at the plasma cell stage (28). Binding sites for the transcription factors SP1 and AP-1/AP-2 are present in the CD21 promoter-containing region (1253/+75), but no regulatory elements that control cell or stage-specific transcription of the CD21 gene have as yet been identified in this region (29). Recently, however, an intronic silencer gene segment (CRS) has been characterized that confers stage-specific expression of a reporter gene under control of the CD21 proximal promoter following stable transfection of B cell lines (30). Although CD21 is not expressed on murine thymocytes, the thymus of mice transgenic for the CRS expressed the reporter gene, suggesting that the construct directs CD21 expression in a human pattern on T cells in this model (30). Such a control of gene expression by an intronic silencer in combination with an active promoter has also been described in thymocytes with regard to CD4 (31).
Proliferation is mostly restricted to the CD4CD8 and early TCR populations of thymocytes (24). IL-7, which is produced by thymic stromal cells, appears to play a major role during early proliferative stages of thymocyte maturation (32). Since CD21 triggers the proliferation of B cells (8), we investigated the effects of CD21 ligation on IL-7-induced proliferation of purified CD21++-expressing early thymocytes. These cells represent high CD21-expressing primary T cells. Soluble/immobilized anti-CD21mAb, iC3b and soluble/cell-associated CD23 were ineffective in modulating the proliferative response of early thymocytes to IL-7. CD23-transfectants used in these experiments were, however, able to cause a 10- to 100-fold reduction in the concentration of anti-IgM required for B cell proliferation (23). Anti-CD21 mAb were also reported to be ineffective in enhancing the proliferation of unfractionated thymocytes to anti-CD2 and/or IL-2 (33). Although Mossalayi et al. ascribed a role for CD23 in prothymocyte maturation (25), we did not observe any phenotypic change upon co-culture of CD21++ thymocytes with CD23-transfected cells, suggesting that CD21 is not the receptor involved in the transduction of a maturation signal. Furthermore, we found no evidence of association of a transducing molecule with CD21 in the membrane of immature thymocytes. In contrast to B cells where CD21 is complexed with the transducing molecule CD19, CD21 was not part of a preformed complex in the membrane of resting thymocytes. The lack of association of CD21 with a potentially transducing protein has also been reported by Prodinger upon analysis of immunoprecipitates of human T cell lines (34). Thymocytestromal cell interaction is a two-way process in which the development and maintenance of stromal cell function is dependent upon the influence of developing thymocytes (35). Since CD23-specific mRNA has been detected in thymic epithelial cells of the outer cortex (36), we suggest that CD21 on early thymocytes acts as a ligand for CD23-expressing cells in the thymus.
The presence of the EBV genome has been reported in several T cell malignancies. The expression of CD21 may thus provide a route for entry of the virus into cells of the T lineage. Suspensions of unfractionated human thymocytes have been reported to be infected by EBV and to allow the transcription of viral genes in the absence of cell immortalization (33,37,38). The dramatic down-regulation of CD21 induced by EBV that we observed on CD21++ thymocytes was likely due to the internalization of the EBVCD21 complex since we found a concommitant decrease in the expression of CD35. CD35 was previously demonstrated to be co-expressed with CD21 in CD4CD8 thymocytes (20) and to co-internalize with CD21 in the HPB-ALL T cell line (19). Intact virions have been detected in membrane-bound cellular vesicles in the CD21-expressing leukemic T cell line HPB-ALL (39). In accordance with a previous report (39), we also provide evidence that infection with EBV up-regulates the CD1a antigen in purified immature thymocytes. EBV-induced phenotypic changes of infected thymocytes may be related to the expression of viral transactivating factors such as ZEBRA (40) which was shown to interfere with cellular transcription factors (41,42).
Taken together, our data provide evidence for a regulated expression of CD21 during intrathymic maturation of human T cells and suggest that the high CD21-expressing subset of immature thymocytes may be a target for infection with EBV. Since the transcription of EBV genes alters the function of RAG which is active in recombination of TCR genes in immature thymocytes (43), our data could be relevant to pathological processes of selection of repertoires in human T cells.
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Acknowledgments
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This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Association pour la Recherche sur le Cancer and Association Franciaise contre les myopathies, France and Biomed-2 program, BMH4-CT96-1005.
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Abbreviations
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DN double negative |
DP double positive |
EBV EpsteinBarr virus |
MFI mean fluorescence intensity |
PE phycoerythrin |
SA streptavidin |
SP single positive |
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Notes
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Transmitting editor: G. Klein
Received 30 April 1999,
accepted 27 July 1999.
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