CD53, a thymocyte selection marker whose induction requires a lower affinity TCRMHC interaction than CD69, but is up-regulated with slower kinetics
Kirsten L. Puls1,
Kristin A. Hogquist2,
Nancy Reilly2 and
Mark D. Wright1,3
1 The Walter & Eliza Hall Institute of Medical Research, Victoria 3050, Australia
2 Center for Immunology, Department of Laboratory medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA
Correspondence to:
M. Wright, The Austin Research Institute, A & RMC, Studley Road, Heidelberg, Victoria 3084, Australia. E-mail: m.wright{at}ari.unimelb.edu.au
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Abstract
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The molecular mechanisms that govern the survival, maturation and export of thymocytes are the subject of intense study, and candidates for involvement in these processes might be identified by their differential expression during thymocyte selection. One such molecule is the tetraspanin CD53, which is not expressed on most CD4+CD8+ double-positive (DP) cells in the normal mouse. We have examined CD53 expression on DP from several class I- and class II-restricted TCR transgenic (Tg) mice, and have found a strong correlation between CD53 expression and positive selection. CD53 expression in DP was formally demonstrated to be dependent upon MHC recognition as evidenced by studying DP from MHC-deficient mice which totally lack expression of this molecule. This link between selection and CD53 expression was reminiscent of CD69, and indeed the majority of selected DP from normal mice that express CD53 also express CD69. We compared CD53 and CD69 induction in vitro using pre-selected thymocytes from TCR-Tg mice that were stimulated either with mAb against TCR or with antigen-presenting cells (APC) pulsed with peptides. The data shows that with either stimulus, CD69 is induced rapidly on the thymocyte surface with expression detected in as little as 2 h. CD53 induction is slower with maximal expression taking up to 20 h. We also stimulated pre-selected thymocytes from the OT-1 TCR-Tg strain with APC pulsed with peptides of varying affinities for the TCR. Here low-affinity peptides which induce CD69 expression poorly were able to induce significant levels of CD53 expression. These data demonstrate that the induction of CD53 and CD69 upon selection is not identical. Thus a combination of the CD69 and CD53 selection markers may be a powerful tool to isolate thymocytes that have either been very recently selected or have arisen from differing MHCTCR affinity interactions during selection.
Keywords: cell surface molecules, FACS, thymus
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Introduction
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CD53 was one of the earliest members of the tetraspanin/transmembrane-4 superfamily to be described (1). Members of this superfamily share a similar structure, consisting of four highly conserved transmembrane domains and two extracellular domains that vary in length and sequence, but which contain strictly conserved cysteine motifs. The conservation of tetraspanins across diverse species and the fact that they are expressed on a wide range of tissues suggests a general function applicable to many cell types. Although roles for these molecules remain largely undefined, a few general features common to most family members have emerged (reviewed in 2,3). CD53 is typical of the superfamily in most of these respects. It is among several tetraspanins to be expressed differentially on cells at different stages of growth and differentiation (46) with CD53 RNA levels increasing upon activation of lymphocytes (7,8). Many tetraspanins form complexes with other surface molecules, including other tetraspanins. CD53 has been shown to participate in a series of complexes with other leukocyte surface molecules, including CD2, CD19, CD21 and MHC class II, and other tetraspanins, including CD37, CD81 and CD82 (9,10). Association between tetraspanins and integrins is emerging as a theme in the recent literature (for review, see 11), and this association may be linked to the ability of tetraspanins such as CD81 and CD9 to influence cell migration and adhesion. CD53 has itself been found to associate with the ß1 integrin, VLA-4 (12), raising the possibility that it may also influence cell motility. Antibody cross-linking experiments have indicated that tetraspanins including CD53 may carry out their functions by influencing signal transduction pathways and may modulate the signaling of other surface molecules with which they associate (reviewed in 2,3). In particular, CD53 is among several tetraspanins capable of co-stimulating Jurkat cells (13), and has been implicated in a variety of signaling effects on leukocytes and cell lines. These include mitogenic stimulation of rat T cells (9), induction of both protein kinase C-dependent and -independent signals (9,14,15), tyrosine phosphorylation events (9,16) including tyrosine kinase-dependent B cell activation (14), and a non-covalent association with a tyrosine phosphatase(s) (17).
Although CD53 is expressed on virtually all peripheral leukocytes, its expression in the thymus is developmentally regulated. In the mouse and rat, CD53 is present on CD4-CD8- double-negative (DN) cells but disappears as they transit to double-positive (DP) via immature single-positive (SP) cells. At the DP stage, when positive selection occurs, CD53 is detectable in only a small proportion of cells, although it is expressed on mature SP cells (1,18,19). This down-regulation of CD53 on cells auditioning for selection suggested that the absence of CD53 might be necessary for the success of the process and perhaps might also be required by newly selected cells. A preliminary study examining the relationship between CD53 and positive selection showed that there might be a correlation between CD53 expression on DP and positive selection in F5 MHC class I-restricted TCR-Tg mice (18). Thymocyte selection is a complex process incompletely understood, and is likely to involve multiple binding of thymocyte TCR to MHC and peptides on thymic stromal cells (reviewed in 20). The avidity of this interaction then determines the fate of the cell (reviewed in 21). However, much remains unknown about the molecular mechanisms that connect the TCR selection signal with different selection fates. The classical positive selection marker CD69, (22), Notch, CD5, CD45 and TCR itself are molecules that are up-regulated upon selection, while Rag-1, Rag-2 (23) and CD43 (24) are down-regulated. Through work with genetically altered mice, the role of some of these molecules has begun to be defined and many of these differentially expressed molecules do influence selection (2528).
In this paper, we demonstrate that CD53 is up-regulated in response to positive selection signals in a number of different class I- and class II-restricted TCR-Tg mice. We also compare the induction of CD53 in DP to the classical selection marker CD69. Our data demonstrate clear differences with regard both to the affinity of the TCRMHC interactions required for their induction and in the kinetics of their expression.
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Methods
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Mice
The class I MHC-restricted TCR-Tg mice used in these analyses were: the H-2Db-restricted male H-Y antigen-specific TCR-Tg mice (29) on both the positively selecting H-2b and non-selecting H-2d backgrounds, and also on SCID and non-SCID backgrounds; OT-I mice [H-2Kb-restricted ovalbumin (OVA)-specific TCR transgene] on the H-2b positively selecting and bm1 or TAP0 non-selecting backgrounds, and also on rag-10 and non-rag-10 backgrounds (30) which were a gift from Dr Bill Heath (The Walter and Eliza Hall Institute); and P14 mice, expressing an LMCV-H-2Db-specific TCR transgene on positively selecting H-2b and non-selecting H-2d backgrounds (a gift from Dr Richard Boyd, Monash University) (31). Class II MHC-restricted TCR-Tg mice used were: OT-II mice, expressing an OVA I-Ab-specific TCR transgene on a positively selecting H-2b background (32), which were a gift from Dr Bill Heath (The Walter and Eliza Hall Institute); HA mice, expressing a hemagglutinin I-Ek-specific transgene on H-2k background (33); and DO11-10 mice expressing an OVA I-Ad-specific TCR transgene on an H-2d background (34). Other mouse strains used were ß2-microglobulin-deficient (35) and MHC II-deficient mice (a gift from Dr F. Koentgen, The Walter and Eliza Hall Institute) (36); MHC double-deficient mice which were a kind gift of Professor Christophe Benoist and Professor Diane Mathis (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France) (37). All mice were maintained in a specific pathogen-free environment at the Walter and Eliza Hall Institute and were used at 38 weeks of age.
Antibodies and peptides
Antibodies used to stain thymocytes and splenocytes included anti-CD4phycoerythrin (PE) GK1.5 (Becton Dickinson, San Jose, CA), and secondary Cy5 and PEstreptavidin conjugates from PharMingen (San Diego, CA). In addition, antibodies conjugated in this laboratory were used, including Cy5- and Texas Red-conjugated anti-CD8 YTS169.4 (38), FITC- and Texas Red-conjugated anti-CD3 KT3-1.1 (39), Cy5 and biotinylated anti-CD53 OX80 and OX79 (18), and FITC-conjugated and biotinylated anti-CD69 H1.2F3 (40). Unconjugated and biotinylated anti-H-Y TCR
chain (T3.70) (41) were a gift of Dr Andreas Strasser (Walter and Eliza Hall Institute). B20.1 (antiV
2) was a gift from Dr Bill Heath (Walter and Eliza Hall Institute). The isotype control antibody used for CD53 was the anti-CD25 mAb 7D4, a rat anti-mouse IgM (42) conjugated to Cy5. In some experiments unconjugated hybridoma supernatants of OX79 and 7D4 were used. Here, anti-rat pan-Igbiotin (Amersham, Little Chalfont, UK) or anti-rat µ chain-specific FITC conjugates (Cappel, Turnhout, Belgium) were used as secondary reagents. The sequences of the Kb-binding peptides used in the work are as follows: OVAp (SIINFEKL), N6 (SIINFNKL), G4 (SIIGFEKL), E1 (EIINFEKL), V-OVA (RGYNYEKL) and P815p (HIYEFPQL). The peptides were synthesized using standard Fmoc chemistry by Research Genetics (Huntsville, AL).
In vitro stimulation of thymocytes
Thymocytes from H-Y H-2Dd or OT-I H-2Kbm-1 non-selecting mice on a non-SCID background were plated out at 5x106 cells/ well on plates coated with either T3.70 (anti-
chain of H-Y TCR) or B20.1 (anti-
chain of H-Y TCR) at 0.5 mg/ml. Alternatively, thymocytes from OT-I TAP0 mice were plated with 1x105 Kb antigen-presenting cells (APC) and various concentrations of peptide (43). Cells were cultured at 37°C for various times, harvested and stained for CD69, CD53, CD4 and CD8.
Staining and flow cytometry
Single cells suspensions were made from thymi of TCR-Tg, MHC knockout or normal CBA/CaH mice. For analyses, 3x106 cells were stained per sample with antibodies at the appropriate concentrations as previously described (44). The cells were resuspended just prior to analysis in medium with propidium iodide at 1 µg/ml, so propidium iodide+ dead cells were removed by gating. Samples were analyzed on FACStar plus or FACScan (Becton Dickinson) flow cytometers, that had been calibrated using CaliBRITE beads. File sizes ranged from 5000 to 200,000 events. FACS data files were analyzed using the Weasel program (Dr F. Battye, Walter and Eliza Hall Institute).
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Results
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CD53 expression in DP thymocytes is induced by positive selection.
We analyzed CD53 expression in DP from a panel of class I- and class II-restricted TCR-Tg mice and MHC knockout mice. Thymocytes were stained with antibodies against CD53, CD3, CD4 and CD8, and analyzed by FACS. Thymocytes of mice expressing the class I-restricted H-Y, OT-I and P14 TCR-Tg were examined either in the presence or absence of the appropriate positively selecting MHC. The histograms in the first three columns of Fig. 1(B)
show that an increased proportion of DP from MHC class I-restricted TCR-Tg mice are CD53+ as compared to DP from TCR-Tg mice on a non-selecting MHC background. The TCR complex is also up-regulated during positive selection (31) and can itself be used as a positive selection marker. Therefore, dividing DP into CD3lo and CD3inthi cells (Fig. 1C
) differentiates cells that are pre- or non-selected from positively selected cells. The first three columns of Fig. 1(D)
show that CD53 is up-regulated on CD3hi DP compared to CD3lo DP from all class I-restricted TCR transgenic mice. Thus the higher proportion of CD53+ DP seen in these mice compared to mice with a non-selecting MHC is probably due to its up-regulation on positively selected cells. Similar results were obtained in the analyses of positive selection of SP CD4 by examining the MHC class II-restricted TCR-Tg mice HA TCR-Tg (I-Ek), DO-11.10 (I-Ad) and OT-II (I-Ab) (Fig. 1
, columns 46). MHC class II-restricted TCR-Tg mice on a non-selecting MHC background were not available, so an isotype control (an anti-CD25 IgM 7D4) was used for comparison. The results show an increased expression of CD53 in the DP of all three MHC class II-restricted TCR-Tg mouse strains, as compared to normal mice (18). Moreover, CD53 is likely to be up-regulated on positively selected DP in these mice, since it is found at high levels on CD3hi DP compared to CD3lo DP (Fig. 1D
). To confirm that the CD53 up-regulation seen on positively selected DP can be driven by selection on both MHC class I and II, thymocytes from MHC knockout mice were also stained with antibodies against CD53, CD3, CD4 and CD8. The MHC knockout mice shown in Fig. 2
are not TCR transgenic, and therefore the percentage of thymocytes with up-regulated levels of CD53 expression is, as expected, low and comparable to levels observed in the DP of normal mice (18). Nonetheless, Fig. 2(B)
shows selection on class I in MHC class II-deficient mice leads to CD53 up-regulation on DP. In the absence of MHC class I in ß2-microglobulin-deficient mice, CD53 is still up-regulated on TCRhi DP, which is further evidence that CD53 is up-regulated on MHC class II-mediated selection. To demonstrate that the appearance of CD53 on DP is the direct result of an MHC-mediated signal, mice deficient for both class I and II were also examined. DP from such mice will not have received any MHCTCR selection signal and should all be pre-selected cells, which is demonstrated by their failure to express CD69 (37). Figure 2(B)
shows that the DP from MHC-deficient mice are clearly negative for CD53 and thus we conclude that a selection signal is required for CD53 to be expressed on DP cells.

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Fig. 1. The expression of CD53 on gated DP from a panel of TCR-Tg mice. Thymocytes were stained with anti-CD3, -CD4, -CD8 and -CD53 antibodies. (A) DP are gated as shown in the CD4 versus CD8 dot-plots for each mouse. (B) Histograms show CD53 expression on DP from each mouse. The background of an isotype control for CD53 (7D4, broken line), or in the case of the class I-restricted TCR-Tg mice (OT-1, H-Y and P-14) CD53 expression on DP from mice with the same TCR transgenes but with non-selecting MHC (broken line), is overlaid for comparison. (C) Histograms showing CD3 expression on gated DP from each mouse. Gates defining CD3hi and CD3lo DP are shown. An isotype control (broken line), or in the case of the class I-restricted TCR-Tg mice (OT-1, H-Y and P-14) CD3 expression on DP from mice with the same TCR transgenes but with non-selecting MHC (broken line), is overlaid for comparison. (D) Histograms showing CD53 expression in CD3hi and CD3lo DP from each mouse. Results are representative of at least four repeated experiments.
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Fig. 2. The expression of CD53 on gated DP from MHC knockout mice. Thymocytes were stained with anti-CD3, -CD4, -CD8 and -CD53 antibodies. (A) DP are gated as shown in the CD4 versus CD8 dot-plots for each mouse. (B) Histograms show CD53 expression on DP from each mouse. The background of an isotype control for CD53 (7D4, broken line) is overlaid for comparison. (C) Histograms showing CD3 expression on gated DP from each mouse. Gates defining CD3hi and CD3lo DP are shown. An isotype control (broken line) is overlaid for comparison. (D) Histograms showing CD53 expression in CD3hi and CD3lo DP from each mouse. Results are representative of at least three repeated experiments.
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CD69 and CD53 are co-expressed on positively selected cells
CD69 and CD53 are apparently markers for positive selection, so presumably they will be expressed on the same cells. However, it is possible that they may delineate subsets of DP that differ in their selection origins, as a result of the quality or quantity of the signal received. We initially addressed this question by staining thymocytes from normal mice with antibodies against CD4, CD8, CD53 and CD69. DP cells were gated and examined for CD53 and CD69 expression. Figure 3(A)
shows that the vast majority of DP in normal mice that are positive for CD53 are also positive for CD69 and that DP which express only one or other of the selection markers are minor populations. However, a possible difference in CD53 and CD69 expression in DP was first suspected when the same analysis was performed on our panel of TCR-Tg mice. The majority of the TCR-Tg mice also show a concordance of CD53 and CD69 expression in DP (data not shown). However, Fig. 3(B)
shows the induction of CD69 in DP from H-Y TCR-Tg mice with positively selecting MHC was poor when compared to the other TCR-Tg mice with positively selecting MHC. This poor induction of CD69 in the H-Y TCR-Tg mice with positively selecting MHC was in contrast to the relatively strong induction of CD53 observed in the DP of these mice (Fig. 1
).

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Fig. 3. CD69 expression in DP from normal and TCR-Tg mice. (A) Examination of CD53 and CD69 co-expression on gated DP from normal CBA/H mice. Thymocytes were stained for CD69, CD53, CD4 and CD8, and CD4+CD8+ DP were gated as indicated. The dot-plot shows CD53 versus CD69 expression on gated DP. Results shown are representative of at least three experiments. (B) CD69 expression on gated DP from a panel of class I MHC-restricted TCR-Tg mice with positively selecting MHC. Thymocytes were triple stained with anti-CD4, -CD8 and -CD69 antibodies, and DP gated as shown in the CD4 versus CD8 dot-plots for each mouse. Histograms show CD69 expression on DP from each positively selecting mouse. CD69 expression on DP from mice with the same TCR transgenes but with non-selecting MHC (broken line) is overlaid for comparison. Results are representative of at least three repeated experiments.
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DP thymocytes from H-Y mice can express CD69
The poor expression of CD69 on DP from H-Y TCR-Tg mice with positively selecting MHC might be an artifact of this transgenic strain. For example, it is possible that due to the insertion of the transgene this strain might be genetically incapable of high level expression of CD69. To address this possibility, DP thymocytes from H-Y non-selecting mice were stimulated in vitro using monoclonal antibodies specific for the H-Y TCR. Expression of CD69 could be detected in as little as 5 h after stimulation (Fig. 4
) and by 10 h the level of expression was significantly stronger than the levels of CD69 expression observed in the DP of H-Y TCR-Tg mice with positively selecting MHC (Fig. 3
). By contrast, there was little CD53 induction at 5 h and we did not detect any CD53 induction until 10 h of stimulation. Similar results were also obtained by stimulating DP from non-selecting OT-I mice (data not shown). We conclude that given sufficient stimulus, H-Y DP thymocytes are genetically capable of inducing significant expression of CD69. Presumably, the stimulus that occurs in the DP thymocytes of H-Y mice in vivo is of insufficient affinity and/or avidity to induce a high level of expression of CD69. Consequently we decided to examine the effect of varying the affinity of TCRMHC interactions on CD53 and CD69 induction. For this we chose the well-studied OT-I Tg model, where the affinity of the TCR for positive and negative selection peptide ligands is known (45,46).

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Fig. 4. Induction of CD53 and CD69 in DP thymocytes from H-Y TCR-Tg mice stimulated in vitro with mAb. (A) Dot-plot shows CD4 and CD8 expression on thymocytes from H-Y H-2Dd mice. (B and C) Histograms show CD53 or CD69 expression on gated CD4+CD8+ cells stimulated for 5 (B) or (C) 10 h with T3.70. The overlay (broken line) is CD69 or CD53 expression on unstimulated gated CD4+CD8+DP. Results are representative of at least three repeated experiments.
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Stimulation of OT-I DP thymocytes with APC and altered peptide ligands.
We examined the effect of varying the affinity and avidity of TCRMHC interactions on CD53 and CD69 induction using an in vitro system where APC are pulsed with peptide and used to stimulate pre-selected DP from OT-I TCR-Tg mice, on a Tap-1-deficient background. Figure 5(A)
shows DP thymocytes that are stimulated with the antigenic peptide OVAp. With this high-affinity antigen peptide, we could detect no significant difference in CD53 and CD69 induction. Both were readily induced, even at low peptide doses.

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Fig. 5. Induction of CD53 and CD69 on OT-I thymocytes in response to various peptide ligands. (A) Thymocytes from OT-I TAP0 mice were stimulated in vitro with APC pulsed with various concentration of the antigenic OVAp peptide or (B) with 20 µM variant peptides. After 20 h, the cells were analyzed for surface expression of CD53 and CD69 on DP thymocytes. Data are expressed as normalized mean fluorescent intensity (MFI), wherein the maximal expression of each protein after induction with 500 nM OVAp is considered 1.0 and the expression level in the absence of stimulation is 0. Results are representative of at least three experiments.
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In Fig. 5(B)
, the effect of varying the affinity of the TCRMHC interaction was examined using altered peptide ligands. The affinity of the OT-I TCR for various Kbpeptide complexes has been measured using Biacore technology (45,46). The peptides N6, G4, E1 and V-OVA are variants of the OVAp peptide, and are listed left to right according to decreasing affinity in Fig. 5(B)
. The control Kb-binding peptide, P815p, did not induce significant CD53 and CD69 expression, as expected. The agonist N6 induced high levels of both CD53 and CD69. However, the G4, E1 and V-OVA peptides induced a relatively higher CD53 expression than CD69. Thus, it would appear that CD53 has a lower threshold than CD69 in terms of the affinity of the interaction required for its up-regulation.
In Fig. 4
, where CD53 and CD69 expression was induced by stimulation of H-Y DP with mAb, we noticed a kinetic difference in CD53 and CD69 induction. At the earlier 5-h time point, we detected CD69 up-regulation in the relative absence of CD53 up-regulation. To further examine whether there was a kinetic difference between CD53 and CD69 induction, we stimulated pre-selection thymocytes from OT-1 TAP0 mice, with APC pulsed with OVAp, and examined CD53 and CD69 expression at varying time points (Fig. 6
). Significant CD69 expression was induced in as little as 2 h, although expression increased with time. By contrast, CD53 expression is marginal until after 20 h of APCthymocyte interaction. This is also true of peripheral lymph node T cells (data not shown). Furthermore, with the low-affinity peptides N6 and G4, which induced moderate CD69 by 24 h, up-regulation was noted as early as 2 h (data not shown), while the CD53 up-regulation was noted later (20 h). Thus, we conclude that CD69 is up-regulated with significantly faster kinetics than CD53, even in situations where its induction is not maximal.

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Fig. 6. Kinetic analysis of CD53 and CD69 induction in DP thymocytes from TAP-deficient OT-I mice stimulated with APC pulsed with OVAp. Thymocytes from OT-I TAP0 mice were stimulated in vitro with APC pulsed with 500 pM OVAp peptide for various periods of time (hrs), as indicated. Results are representative of at least three experiments.
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Identification of CD69+ CD53- DP in vivo
The data from in vitro stimulation of DP thymocytes points to differences in the induction of CD53 and CD69. CD53 expression can be induced by low-affinity TCRMHC interactions which fail to induce significant levels of CD69 (Fig. 5
) and the existence of CD53+CD69- DP can occur in vivo as demonstrated by the analysis of the H-Y TCR-Tg mice (Fig. 3
). In contrast, CD69 is induced with a markedly quicker kinetics than is CD53 (Figs 4 and 6
), at least in vitro. It is clear that in normal mice the majority of post-selected thymocytes which express CD53 also express CD69 (Fig. 3A
). However, given the differences in the CD53 and CD69 induction observed in vitro, we decided to revisit the issue, and determine whether we could detect and analyze a minor CD69+CD53- DP population in vivo. Five-color FACS analyses was performed on thymocytes from normal mice, using antibodies against CD4, CD8, CD3, CD69 and CD53. We first compared CD53 and CD69 expression, and analyzed the CD69+CD53-thymocytes for CD4 and CD8 expression (Fig. 7a and b
). The majority of CD69+CD53- thymocytes are clearly DP. Strikingly they are CD4lo and CD8loint compared to the majority of DP. We then compared CD3 expression in the CD69+CD53- DP with total DP (Fig. 7c
). The CD69+CD53- DP that have up-regulated their CD3 expression are CD3lo, consistent with these cells being very recently selected. These data are consistent with a more rapid temporal induction of CD69 than CD53 during positive selection.

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Fig. 7. Thymocytes from a normal mouse stained with CD69, CD53, CD3, CD4 and CD8. (A) A dot-plot of total thymocytes showing CD69 against CD53 staining was gated for CD69+ SP, an isotype control is shown for comparison. (B) Dot-plots showing CD4 and CD8 expression on CD69+CD53- DP. Ungated thymocytes are shown as a control. (C) CD3 expression on CD69+CD53- DP, the overlay (broken line) is CD3 expression on ungated thymocytes. CD3 expression on gated total DP is shown as a control in comparison to an isotype control (overlay broken line). Results are representative of at least four repeated experiments.
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Discussion
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Analyses of three class I-restricted TCR-Tg mice has shown that the proportion of CD53+ DP is significantly increased in TCR-Tg mice expressing a positively selecting MHC as compared to TCR-Tg mice with non-selecting MHC (Fig. 1
). Since CD53 is present on both CD4 and CD8 SP, it was expected that CD53 would also be found to be up-regulated upon class II-selected cells, although some molecules such as F3Ag are down-regulated specifically in one co-receptor lineage but not the other (47). Examination of CD53 expression in class II-restricted TCR-Tg mice indeed revealed a population of CD53+ DP, although this appeared smaller than had been seen in most class I-restricted TCR-Tg mice (Fig. 1
). However, a comparison of CD53 expression on CD3hi and CD3lo DP in class II-restricted TCR-Tg mice shows a marked up-regulation in the selected CD3hi DP population. Moreover, examination of ß2-microglobulin knockout mice showed that in the absence of class I MHC, CD53+ DP are still present and analyses of MHC double-deficient mice confirmed that CD53 expression on DP thymocytes is dependent on an MHC-mediated signal, since CD53 is not present on DP from these mice (Fig. 1
). Thus, CD53 is up-regulated on the selected precursors of both co-receptor lineages at the DP stage. Non-selected and pre-selected DP apparently do not express CD53 (Fig. 2
) (18).
It is interesting to note the variation observable in CD53 expression among the different strains of TCR-Tg mice. Firstly, in the class I-restricted TCR-Tg mice, CD53 up-regulation was modest in P14 mice (data not shown but similar to what was seen in the F5 mice) (18), but was much greater in OT-I and H-Y mice (Fig. 1
). Given that the affinities for all four class I-restricted TCR thus far examined have not been accurately determined, we cannot rule out the possibility that the varying induction of CD53 during selection may reflect the strength of TCRMHC interactions, and may be influenced by such variables as TCR affinities and density of TCR expression. Secondly, the induction of CD53 in the class II-restricted TCR-Tg mice was modest as compared to the class I-restricted OT-I and H-Y mice (Fig. 1b
). Again, there is a possibility that this modest up-regulation in the proportion of CD53+ DP in class II-restricted thymocytes reflects the strength of MHCTCR interactions and perhaps a more impressive induction of CD53 may be observed upon examination of further class II-restricted TCR-Tg strains. Alternatively, there is evidence from a number of different experimental systems that cells selected on class II MHC remain in the DP compartment for a shorter time then the cells selected on class I MHC (4850). Thus, class II selected cells may down-regulate CD8 before they have fully up-regulated CD53, hence a greater accumulation of CD53+ cells is observed in class I-restricted TCR-Tg mice compared to class II-restricted TCR-Tg strains.
CD69 is a C-type lectin superfamily protein that exists as a dimer and is the classical marker of thymocyte positive selection (reviewed in 22). It is not found on most resting peripheral leukocytes, but like CD53 (data not shown) is up-regulated on activation. It is also like CD53 in that it is able to contribute to signal transduction in leukocytes. Given that both CD53 and CD69 are both markers for selected DP thymocytes, we compared their induction in DP thymocytes in vitro where we could readily control the duration and affinity of the TCRMHC interactions. Experiments from two different systems suggest that given a sufficient signal, CD69 is up-regulated with a significantly faster kinetics than is CD53. Firstly, pre-selected DP expressing the H-Y TCR transgene were induced with mAb against TCR within 5 h to express CD69 at their surface. At this early time point, no significant CD53 expression was observed (Fig. 4
). Similarly, pre-selected DP expressing the OT-1 TCR transgene could be induced by the stimulus of APC and OVAp to express significant levels of CD69 in as little as 2 h. An equivalent level of CD53 expression was not observed until 20 h (Fig. 6
).
The stimulation of pre-selected DP OT-1 thymocytes using altered peptide ligands showed a clear difference in the signal threshold requirements for CD53 and CD69 induction. Both molecules were readily induced by OVAp, a high-affinity peptide which can stimulate mature peripheral OT-1 T cells in vitro and which generally induces negative selection in fetal thymic organ culture. By contrast, the antagonist V-OVA peptide, which has a 5-fold lower affinity for OT-I TCR than does OVAp (46), and which fails to stimulate proliferation of peripheral OT-1 T cells, failed to induce either molecule. The most interesting results were obtained when the pre-selected OT-I DP were stimulated with peptides whose affinity was intermediate between V-OVA and OVA. N6 is a strong OT-I agonist. Yet this peptide, of lower affinity than OVAp which induced CD53 expression well, induced a somewhat weaker expression of CD69 than did OVA. The results were more striking for G4 and E1. These partial agonists are both powerful inducers of positive selection. Here, they induce CD69 poorly despite a strong induction of the CD53 marker. Thus, the data from the in vitro system clearly point to two major differences in the induction of CD53 and CD69 following interactions of DP with selecting MHC. Firstly, CD53 can be induced in response to lower-affinity MHCTCR interactions than CD69. Secondly, given an appropriate stimulus, CD69 is induced with a significantly quicker kinetics than CD53.
The question raised by this in vitro data is whether the differing requirements for CD53 and CD69 induction in DP is reflected in the existence of selected DP populations whose expression of the two selection markers differs. Examination of the DP from H-Y mice with positively selecting MHC showed that the expression of CD69 and CD53 can differ in selected DP in vivo. The expression of CD69 in the DP of positively selecting H-Y mice is poor when compared to the panel of TCR-Tg mice, a result in agreement with other workers who reported that only 11% (51) or 20% (52) of TCR+ DP are CD69+ in the H-Y mice. However, given a high-affinity stimulation, such as cross-linking the TCR with clonotypic mAb, DP from the H-Y mice are capable of expressing significant levels of CD69 (Fig. 5
). Although the affinity of the H-Y TCR for its antigenic peptide and its selecting MHC has not, to our knowledge, been measured, it has been suggested by several groups to be relatively low (5356). Thus, a likely explanation for this relatively poor CD69 induction in DP from H-Y mice with positively selecting MHC is that the avidity with which the H-Y TCR recognizes its selecting peptide(s)MHC in the thymus is relatively low, and perhaps similar to the avidity of the interaction of the OT-I TCR with its partial agonist peptides G4 and E1. Thus, given this relatively low-affinity TCRMHC interaction, CD53 is induced in the absence of a strong CD69 expression. Thus CD53+CD69- DP can exist in vivo and one might predict that CD69+CD53- DP also exist. The former population would represent DP that have been selected as a result of lower-affinity TCRMHC interactions and that these DP have received an insufficient signal to up-regulate CD69. On the other hand, CD69+CD53- DP might represent very recently selected DP that have received a relatively high-avidity signal and have not yet up-regulated CD53. Figure 2
showed that the vast majority of selected DP from normal co-express CD53 and CD69. Thus, any low-affinity signals in normal mice which are able to induce expression of only CD53 or a transient CD69+CD53- DP that is yet to up-regulate CD53 must be very rare. However, a five-color FACS analyses of normal thymocytes showed that CD69+CD53- DP can be detected. Moreover, given that the earliest DP are thought to be CD3loCD4loCD8lo (57), the phenotype of the CD69+CD53- DP (Fig. 7
) is highly suggestive of a relatively immature, very recently selected population. Isolation of this population using both the CD69 and CD53 selection markers might prove a useful approach to study the changes in gene expression which occur in the early stages of thymic selection.
The absence of the peripheral leukocyte marker CD53 from thymocytes undergoing the earliest phase of selection suggests that the signal or function it elaborates will be inappropriate for cells at this stage of development. We have shown that the thymic positive and selection signal is able to cause up-regulation of CD53. It is of course possible that this up-regulation of CD53 expression during T cell selection is entirely coincidental. However, there are two potential mechanisms by which CD53 may make a functional contribution to T cell development. The first is based on the observations that CD53 can couple to signal transduction pathways (9,1416). Thus, CD53 might actively contribute to signaling in thymocytes, perhaps to the selection signal itself. The selection signal is complex and leads to many outcomes including survival or death, lineage commitment and differentiation. It is thought to be composed not just of a single event, but a series of signals, so that sustained MHCTCR contact is required for successful selection (50,52). There is also evidence that thymocyte responses to TCR signals are progressively altered as they mature (43,58) Thus, although molecules like CD53 appear as a result of the selection signal, it still might be a molecule able to modulate further signals.
A second possible mechanism by which CD53 may contribute to T cell development arises from evidence that tetraspanins influence cell migration and adhesion via their associations with integrins (11), and an interesting role for CD53 in the thymus is suggested by its ability to complex with integrins such as VLA-4 in human lymphocyte cell lines (12). Thymocyte migration is an important component of T cell maturation (59). CD53 is up-regulated as the DP transit form CD3lo to CD3hi and it is at this stage that thymocytes migrate from the cortex to the medulla. Perhaps CD53 might be involved in the migration of thymocytes from the cortex to the medulla by regulating integrin adhesiveness. Whatever its function in T cell development, CD53 may prove to be a useful marker in future studies of thymocyte selection, particularly in combination with CD69.
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Acknowledgments
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This work was supported by the National Health and Medical Research Council, Australia and the Anti-Cancer Council of Victoria, Australia. We thank Professor Ken Shortman, Dr Wu Li and Dr Bill Heath for discussion during the progression of the work and for critical reading of this manuscript. We also thank David Vremec and Dr Wu Li for technical advice. Dr Bill Heath, Dr Andreas Strasser, Dr Frank Koentgen, Dr Richard Boyd, Dr Ann Chidgey, Dr Alan Harris, Professor Christophe Benoist and Professor Dianne Mathis all kindly provided many of the reagents used in this study.
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Abbreviations
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APC antigen-presenting cell |
DN double negative |
DP double positive |
OVA ovalbumin |
SP single positive |
Tg transgenic |
 |
Notes
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3 Present address: The Austin Research Institute, A & RMC, Studley Road, Heidelberg, Victoria 3084, Australia 
Received 25 July 2001,
accepted 23 November 2001.
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