A potential role for CD69 in thymocyte emigration

Chiguang Feng1, Kenneth J. Woodside1, Barbara A. Vance2, Dalal El-Khoury1, Matilde Canelles3, Jan Lee4, Ronald Gress2, B. J. Fowlkes3, Elizabeth W. Shores4 and Paul E. Love1

1 Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, 2 Experimental Transplantation and Immunology Branch, National Cancer Institute, and 3 Laboratory of Molecular and Cellular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA 4 Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA

Correspondence to: P. E. Love; E-mail: pel{at}helix.nih.gov
Transmitting editor: P. Ohashi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The early activation marker, CD69, is transiently expressed on activated mature T cells and on thymocytes that are undergoing positive or negative selection in the thymus. CD69 is a member of the NK gene complex family of C-type lectin-like signaling receptors; however, its function is unknown. In this report, we describe the characterization of mice that constitutively express high levels of surface CD69 on immature and mature T cells throughout development. Constitutive surface expression of CD69 did not affect T cell maturation, signaling through the TCR or thymocyte selection. However, phenotypically and functionally mature thymocytes accumulated in the medulla of CD69 transgenic mice and failed to be exported from the thymus. The retention of mature thymocytes correlated with transgene dose and CD69 surface levels. These results identify a potential role for CD69 in controlling thymocyte export, and suggest that the transient expression of CD69 on thymocytes and T cells may function to regulate thymocyte and T cell trafficking.

Keywords: CD69 antigen, development, lymphocyte activation, transgenes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte migration is known to play an important role in regulating the localization and orchestration of immune responses. Although less well defined, the processes that regulate trafficking of lymphoid precursors to and within sites that support their development, and that mediate the subsequent emigration of mature cells to the periphery, are also critical for the establishment of cellular immunity. T lymphocyte precursors that originate in the fetal liver or bone marrow migrate to the thymus and subsequently undergo a complex differentiation process that includes positive and negative selection, before emerging as CD4+ or CD8+ single-positive (SP) T cells. As thymocytes progress through these developmental stages, they migrate from the subcapsular region of the thymus to the cortex and then to the medulla (1). At the conclusion of this maturation process, functionally mature thymocytes exit the thymus and seed the peripheral lymphoid tissues.

Results from several recent studies indicate that newly generated SP thymocytes undergo several additional maturation steps before being exported from the thymus (2,3). In addition, fully mature SP thymocytes also display a phenotype that resembles that of recent thymic emigrants (RTE) which is distinct from most other medullary thymocytes (4,5). The mechanisms that control thymocyte emigration and the molecules involved in this process remain ill-defined. A pertussis toxin-sensitive pathway, therefore likely involving Gi-proteins, has been implicated in regulating thymocyte emigration (6,7). Chemokine receptors, which have been shown to regulate migration of mature lymphocytes and which signal through G-protein-mediated pathways, are therefore leading candidates for controlling thymocyte export (8). Adhesion molecules and their signaling pathways are also likely to play important roles in thymocyte emigration (9).

The early activation marker, CD69, is a member of the NK cell gene complex family of C-type lectin-like signal-transducing receptors. CD69 is a type II transmembrane glycoprotein that contains a C-type lectin-binding extracellular domain (1013). Although CD69 is expressed by a wide range of hematopoietic lineages, its function and ligand remain unknown. The timing and pattern of CD69 induction is similar in all cells that express this protein and is characterized by rapid but transient surface expression. In T cells, CD69 is up-regulated very early after TCR triggering and wanes soon after stimulation withdrawal (1417). In the thymus, immature CD4+CD8+ [double-positive (DP)] thymocytes that are undergoing positive or negative selection also transiently express CD69 (1821). However, the lack of a thymocyte or T cell phenotype in CD69–/– mice suggests that it does not play a critical role in T cell maturation (22).

To examine the function of CD69 in T cell development, we generated CD69 transgenic (Tg) mice that constitutively express CD69 at all stages of T cell development. We show here that overexpression of CD69 does not impair thymocyte development, but results in accumulation of functionally mature SP thymocytes in the thymus and a paucity of T cells in the periphery. Our results suggest a role for CD69 in controlling the export of mature cells from the thymus and indicate that transient activation-induced CD69 surface expression may be important for regulating T cell trafficking.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents and antibodies
Fluorochrome-conjugated anti-CD3, -CD4, -CD8, -CD24, -CD69, -CD62L, -ß7 integrin, and biotinylated anti-CD8 and -CD45R (B220) mAb were purchased from PharMingen (San Diego, CA). 5-Bromodeoxyuridine (BrdU) was purchased from Sigma-Aldrich (St Louis, MO). CFSE was purchased from Molecular Probes (Eugene, OR). Allophycocyanin-conjugated CD8 for confocal staining was purchased from Caltag (Burlingame, CA). Alexa Fluor 488-conjugated anti-CD4 was prepared by labeling purified anti-CD4 mAb (PharMingen) using the Alexa Fluor 488 protein labeling kit (Molecular Probes) according to the manufacturer’s instructions.

Mice
CD69 Tg mice were established and maintained in our animal facility under specific pathogen-free conditions. The transgene construct was generated by cloning the CD69 coding region into a vector containing the human CD2 promotor and enhancer. The resulting construct (huCD2–CD69) was linearized and injected into B6 zygotes (23). C57BL/6 and Rag-2–/– mice were purchased from Taconic (Germantown, NY). TCR Tg mice used in these studies included H-Y and AND TCR Tg mice. H-Y mice express an MHC class I-restricted TCR for the male antigen H-Y (24) and AND mice express an MHC class II-restricted TCR specific for pigeon cytochrome c (25). All TCR Tg mice were maintained in the H-2Db background.

Flow cytometry
Cells (1 x 106/sample) isolated from thymus, lymph nodes or spleens were suspended in 50 µl of FACS buffer (1 x HBSS, 0.1% BSA and 0.01% sodium azide) with a cocktail of FITC-, phycoerythrin-, Quantum Red-, Per-CP- or allophycocyanin-conjugated antibodies. The mixtures were incubated at 4°C for 1 h. Unbound antibodies were washed out and cells were resuspended in FACS buffer, and then analyzed by flow cytometry using a FACScan or FACSCalibur instrument (Becton Dickinson, San Jose, CA).

BrdU labeling of thymocytes
BrdU (0.8 mg/ml) was administrated continuously to mice in drinking water. Thymocytes were isolated from treated mice at various time points, and 2 x 106 cells/sample were stained with fluorochrome-conjugated anti-CD4 and -CD8 mAb. After washing out the unbound antibodies, cells were fixed with ethanol and paraformaldehyde (PFA). After DNase I treatment of fixed cells, intracellular staining was performed using FITC-conjugated anti-BrdU antibody (PharMingen). Cells were washed and suspended in PBS for analysis by flow cytometry.

Purification and adoptive transfer of CD4 SP thymocytes
Thymocytes were suspended in 4 x 107/ml PBS containing 1% BSA, and incubated with biotinylated anti-CD8 and -B220 antibodies. After incubation on ice for 15 min, unbound antibodies were washed out with PBS and 10 µl of streptavidin–beads (Miltenyi Biotec, Auburn, CA) was added. After another 15-min incubation, cells were passed through a magnetic separation column (Miltenyi Biotec). The flow-throughs were collected, and the purity of each fraction was checked by staining with CD4 and CD8 antibodies. CD4 SP cells in purified populations were typically >80% from non-Tg mice and >90% from CD69 Tg mice. Purified CD4 SP thymocytes were suspended in PBS (2 x 107/ml) and i.v. injected into Rag-2–/– or C57BL/6 mice. In indicated experiments, purified cells were resuspended in PBS (1 x 107/ml) and stained with 1 µM CFSE for 10 min at 37°C before transfer. Cells were washed twice with PBS and re-suspended in PBS for injection. Cells from indicated organs of recipients were isolated and analyzed with antibody staining at different time points after transfer. Recoveries were calculated by multiplying total cell number by the percentage of CD4 SP or CD4 SP CFSE+ cells in each organ.

Proliferation assay
Single-cell suspensions were prepared from thymi in RPMI plus 10% FCS. CD4 SP thymocytes were enriched by panning with CD8, B220 and Mac-1 antibodies on rabbit anti-mouse IgG-coated plates, followed by a positive selection by magnetic separation using CD4–biotin/streptavidin microbeads in the MACS system (Miltenyi Biotec) as indicated above. Accessory cells and antigen-presenting cells (APC) were prepared from spleen cell suspensions from B10.A mice. APC were depleted of T cells with anti-Thy 1.2 + C' and irradiated with 3000 rad. Then 1 x 105 responder T cells were combined with 5 x 105 accessory cells in flat-bottom 96-well plates in the presence or absence of the indicated stimulants. To determine the dose response, a constant number of APC (5 x 105) was combined with 2-fold dilutions of responder T cells. A peptide of pigeon cytochrome c (fragment 81–104) was synthesized in the FDA Core Facility and added to culture at the indicated concentrations. Following stimulation for 48 h, cells were pulsed for 12 h with 1µCi [3H]thymidine and harvested. Antibodies used for panning, including anti-CD8 (2.43), -B220 (6B2) and -MAC-1 (M1/70), were purified with Protein G from tissue culture supernatant generated from B cell hybridomas grown in an Artificial Capillary System (Cellco, Germantown, MD).

Confocal microscopy
Thymi were fixed with 4% PFA for 10 min before being embedded and frozen in OCT compound (Sakura Finetec, Torrance, CA). Cryostat sections (10 µm) were prepared (Histoserv, Gaithersburg, MD). Sections were washed in PBS, fixed with 4% PFA and quenched with 50 mM NH4Cl before staining. Sections were incubated with Alexa Fluor 488-conjugated anti-CD4 and allophycocyanin-conjugated CD8 at 4°C overnight in the dark. Sections were washed, mounted with Prolong Antifade reagent (Molecular Probes) and dried overnight at room temperature with drierite, and then analyzed by confocal microscopy.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Phenotype of CD69 Tg mice
Four huCD2–CD69 Tg (CD69 Tg) founder lines were generated by zygote micro-injection and analyzed. To assess CD69 transgene expression, we first examined CD69 surface levels on thymocyte subsets by flow cytometry. In non-Tg mice, CD69 is expressed on the surface of a minor population of CD4+CD8+ [double-positive (DP)] and ‘transitional’ CD4+CD8 (CD4 SP) and CD4CD8+ (CD8 SP) thymocytes (2,18). In contrast, in CD69 Tg mice, surface expression of CD69 was observed on all thymocyte subsets (Fig. 1A and C, and data not shown). Moreover, CD69 surface levels paralleled transgene copy number (data not shown). Representative founder lines containing either a low (line #2005) or high (line #2028) transgene copy number were chosen for further investigation.



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Fig. 1. Phenotype of CD69 Tg mice. Thymocytes and lymph node cells isolated from CD69 Tg and non-Tg mice were stained with anti-CD4, anti-CD8 and either control, anti-CD3 or anti-CD69 mAb, and analyzed by flow cytometry. (A) CD69 expression levels on total thymocytes from four founder lines and a non-Tg littermate are shown. (B) CD4 versus CD8 staining profiles of thymocytes (Thy) and lymph node (LN) cells, with representative total cell numbers (N) and percentages of each population shown. CD3 expression levels in total thymocytes (C) and lymph node cells (D), as well as CD69 expression levels on DP (CD4+CD8+) and SP (CD4+CD8 and CD4CD8+) thymocytes, and SP (CD4+CD8, CD4CD8+) lymph node T cells are shown.

 
Constitutive surface expression of CD69 had no apparent effect on early thymocyte development as assessed by the normal distribution of double-negative (DN) thymocyte subsets (based on staining with CD25 and CD44; data not shown) and the presence of normal numbers of DP thymocytes in CD69 Tg mice. However, mice from all CD69 Tg founder lines contained higher percentages and numbers of CD4 SP and CD8 SP thymocytes relative to non-Tg littermates (Fig. 1B). The increased number of SP thymocytes in CD69 Tg mice was consistently observed and was accompanied by a concomitant decrease in the percentage, but not the number, of precursor DP thymocytes (Fig. 1B).

Surprisingly, examination of peripheral lymphoid organs revealed that the accumulation of SP thymocytes in CD69 Tg mice also correlated with a decrease in the number of mature peripheral CD4 SP and CD8 SP T cells (Fig. 1B). The reduction in peripheral T cell numbers also paralleled transgene copy number and was observed in all secondary lymphoid organs examined including lymph nodes, spleen and Peyer’s patches (Fig. 1B and data not shown). Analysis of the phenotype of the few T cells present in lymph nodes of the representative high-copy-number line, #2028, revealed that only ~50–60% of the cells were CD69+ (Fig. 1D, left column). In addition, most T cells were CD62Llow CD44high, suggesting that they may have undergone homeostatic proliferation (data not shown). Similar to {alpha}ß-lineage cells, the number of {gamma}{delta}TCR+ thymocytes was increased, but the number of peripheral {gamma}{delta} T cells was decreased in high-copy-number CD69 Tg mice (data not shown).

In contrast to the results obtained with the high-copy-number Tg lines, normal or only slightly reduced numbers of peripheral T cells were found in low-copy-number CD69 Tg mice. As in non-Tg mice, these cells were predominantly CD69 (Fig. 1D, middle column). Analysis of T cells from the low-copy-number CD69 Tg lines by Southern blotting confirmed that the transgene was still present; however, Tg CD69 mRNA levels were reduced relative to those of total thymocytes from the same mice (data not shown). These data suggest that the absence of CD69 surface expression on most peripheral T cells in low-copy-number Tg mice is most likely due to a reduction in transgene mRNA expression. Indeed, previous data indicate that huCD2-mediated transgene expression decreases in mature T cells relative to immature thymocytes (26). Consistent with this interpretation, we observed that although CD69 surface expression was elevated on DP thymocytes from line #2005 Tg mice, it was similar to that of non-Tg mice on SP thymocytes (Fig. 1C, middle column). This suggested that the reduction in CD69 mRNA levels and CD69 surface expression occurred at the SP stage. The reduction in CD69 expression in SP thymocytes relative to DP thymocytes was also seen in the high-copy-number Tg lines, although most SP thymocytes still expressed CD69 (Fig. 1C, left column; data not shown).

Kinetics of thymocyte development in CD69 Tg mice
To determine if the increase in SP thymocytes in CD69 Tg mice was due to a faster maturation rate, we examined the kinetics of thymocyte development by analyzing thymocyte subsets following continuous BrdU oral administration. BrdU is incorporated into proliferating cells, which in the thymus are predominantly late DN thymocytes, and these cells remain BrdU+ during subsequent maturation stages (27,28). As shown in Fig. 2(left panels), the kinetics of DP thymocyte labeling were similar in non-Tg and CD69 Tg mice as assessed by the absolute number and percentage of BrdU+ DP thymocytes observed at different time points. Similar numbers of BrdU+ CD4 SP thymocytes were also detected in CD69 Tg mice and non-Tg littermates, indicating that SP thymocytes are generated at the same frequency in these mice (Fig. 2A, right panel). However, the percentage of BrdU+ cells among all CD4 SP thymocytes was consistently lower in CD69 Tg mice relative to non-Tg littermates (Fig. 2B, right panel). The time required to label 50% of all CD4 SP thymocytes (t1/2) was ~6 days in non-Tg mice, consistent with previous reports indicating that the average intrathymic ‘lifespan’ of SP thymocytes is 12 days (29). However, in CD69 Tg mice, the t1/2 was prolonged to at least 12–14 days, indicating that SP thymocytes remain in the thymus for a much longer time than in non-Tg littermates (Fig. 2).



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Fig. 2. Kinetics of thymocyte development in CD69 Tg mice. CD69 Tg mice (6–10 weeks old, line #2028) and their non-Tg littermates were continuously administrated BrdU (0.8 mg/ml) through their drinking water. Mice were sacrificed at different time points, and the number and percentage of BrdU+ thymocytes was determined. Numbers (A) and percentages (B) of BrdU+ cell in the CD4+CD8+ (left panels) and CD4+CD8 (CD4+) (right panels) compartments are shown. At least three mice were used for each time point.

 
Thymocyte selection is unaffected in CD69 Tg mice
DP thymocytes that receive activating signals in the thymus by interacting with positively or negatively selecting ligands transiently express CD69 (1821). To determine if the increase in SP thymocytes in CD69 Tg mice was due to an alteration in thymocyte selection, we bred TCR transgenes into the CD69 Tg background and examined the efficiency of thymocyte selection. The MHC class I-restricted TCR transgene, H-Y, promotes positive selection of large numbers of CD8 SP thymocytes in female mice and strong negative selection of thymocytes in male mice (30,31). Comparison of H-Y TCR Tg x CD69 Tg and H-Y TCR Tg male mice revealed no difference in the efficiency of negative selection (Fig. 3B). In addition, accumulation of CD8lowCD4 cells in the thymus and their paucity in the periphery was observed in H-Y TCR Tg x CD69 Tg just as in non-TCR Tg x CD69 Tg mice (Fig. 3B). In order to distinguish the effect of constitutive CD69 expression on thymocyte positive selection from accumulation of SP thymocytes, we chose to analyze the low-copy-number CD69 Tg line, #2005, to assess positive selection. In this line, CD69 expression is elevated on all DP thymocytes, but only a slight accumulation of SP thymocytes is observed in non-TCR Tg mice (Fig. 1B). Examination of H-Y TCR Tg x CD69 Tg females revealed no significant difference in the efficiency of positive selection relative to H-Y TCR Tg littermates (Fig. 3A). We also observed no difference in positive selection when the MHC class II-restricted TCR transgene AND was tested (data not shown). Finally, the extent of activation-induced death of DP thymocytes in response to CD3 plus CD28 stimulation was similar in CD69 Tg mice and non-Tg littermates (data not shown).



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Fig. 3. Positive and negative selection in CD69 Tg mice. CD69 Tg mice were mated to HY-TCR Tg mice. Thymocytes and lymph node cells, isolated from double-Tg or HY-TCR Tg female (A) and male (B) mice, were stained with mAb and analyzed by flow cytometry. Total cells numbers (N), and CD4 and CD8 profiles with percentage of each subset are shown.

 
Accumulation of mature SP thymocytes in high-copy-number CD69 Tg mice is due to failure of thymocyte emigration
To explore further the reason for the accumulation of SP thymocytes in high-copy-number CD69 Tg mice, we analyzed thymocyte profiles from mice beginning at birth when the first wave of mature SP cells is observed in the thymus. There was no significant difference in the percentage of SP thymocytes between CD69 Tg mice and non-Tg littermates on day 1, again indicating that the rate of SP thymocyte formation was not increased in CD69 Tg mice relative to non-Tg littermates (Fig. 4A). However, increased numbers and percentages of SP thymocytes were observed in CD69 Tg mice by day 5 and were consistently seen thereafter (Fig. 4A). SP T cells were detectable in spleens of non-Tg mice on day 5 and on all subsequent days, whereas peripheral T cells were first detected in low numbers only on day 10 or later in CD69 Tg mice (Fig. 4B). These results indicated that constitutive expression of CD69 either inhibits the complete maturation of SP thymocytes or inhibits export of SP thymocytes from the thymus.



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Fig. 4. Effect of CD69 transgene on postnatal T cell development. CD69 Tg mice (line #2028) and non-Tg littermates were sacrificed 1, 5, 11 and 15 days after birth, and their thymocytes (A) and splenocytes (B) were analyzed by flow cytometry. Numbers indicate the percentage of cells in each compartment. Average thymocyte numbers (N) and numbers of mice analyzed are shown.

 
Development of newly generated SP thymocytes into functionally mature T cells is a multi-step process that occurs as these cells migrate through the medulla (2,3). Thus, the accumulation of SP thymocytes in CD69 Tg mice could be due to failure of these cells to complete the maturation process. The phenotype of fully mature SP thymocytes has been previously established by examining the phenotype of RTE (4). RTE display phenotypes distinct from most other medullary thymocytes: CD4 SP RTE are CD24low, CD62Lhigh ß7 integrinint whereas CD8 SP RTE are CD24low, CD62Lhigh, CD45RBhigh ß7 integrinhigh. Analysis of the percentage of RTE-phenotype CD4 SP and CD8 SP thymocytes revealed an ~2- to 3-fold increase in these subsets in CD69 Tg mice relative to non-Tg littermates (Fig. 5A). These results correlate with the 2- to 3-fold increase in total SP thymocyte subsets (Fig. 1B), indicating that most of the accumulated SP thymocytes in CD69 Tg mice are phenotypically mature. To determine if the SP thymocytes in CD69 Tg mice were functionally mature, CD4 SP cells from AND TCR Tg and AND TCR Tg x CD69 Tg mice were stimulated with varying concentrations of agonist peptide in the presence of APC. As shown in Fig. 5(B), the proliferative response of CD4 SP thymocytes was significantly greater with cells from AND TCR Tg x CD69 Tg mice relative to AND TCR Tg mice (Fig. 5B). To obtain an estimate of the number of functionally mature cells, the proliferation assay was repeated with serial dilutions of purified CD4 SP thymocytes from CD69 Tg and non-Tg mice. In agreement with the phenotypic analysis, these data indicated that AND TCR Tg x CD69 Tg mice contained approximately twice the number of functionally mature CD4 SP thymocytes as non-CD69 Tg/AND TCR Tg littermate controls (Fig. 5B, right panel). Taken together, these results indicate that constitutive expression of CD69 does not interfere with thymocyte development, but does inhibit the export of functionally mature SP thymocytes to the periphery.



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Fig. 5. Accumulation of phenotypically and functionally mature SP thymocytes in CD69 Tg mice. Thymocytes isolated from CD69 Tg (line #2028) mice and non-Tg littermates were stained with anti-CD62L, -ß7 integrin or -CD44, and anti-CD4 and -CD8 mAb, and analyzed by flow cytometry to determine percentages of cells with the RTE phenotype. Percentages (A) of CD4+CD8 (CD4+) (left panels) and CD4CD8+ (CD8+) (right panels) thymocytes with the RTE phenotype are shown. (B) Ability of CD4+ thymocytes from CD69 Tg AND Tg and AND Tg mice to proliferate in response to pigeon cytochromic peptide. Peptide dose (left panel) and responder cell number (right panel) versus thymidine incorporation are shown. For the experiment shown in the left panel, 1 x 105 CD4+ cells were used. For the experiment shown in the right panel, the peptide concentration used was 60 µM.

 
Constitutive expression of CD69 does not affect mature T cell survival
In addition to a block in SP thymocyte emigration from the thymus, the paucity of T cells in lymph nodes and spleen of high-copy-number CD69 Tg mice could also be due to abnormal migration in the periphery or to the rapid death of thymic emigrants. To examine these possibilities, we adoptively transferred purified CD4 SP thymocytes from non-Tg or line #2028 CD69 Tg mice into Rag-2–/– recipients by i.v. tail injection. The recovery of donor cells from CD69 Tg mice 3 or 7 days after transfer in Rag-2–/– recipient spleens was comparable with that from non-Tg littermates (Fig. 6A). Few donor cells were detected in the thymus of Rag-2–/– recipients 7 days after transfer indicating that CD4 SP thymocytes from CD69 Tg mice do not preferentially return to the thymus (Fig. 6A). Analysis of donor CD4 SP thymocytes that had been pre-labeled with CFSE revealed that cells from CD69 Tg mice underwent homeostatic proliferation in Rag-2–/– recipients to a similar extent as non-Tg thymocytes, even though they remained CD69+ (Fig. 6B). We also investigated the tissue localization of adoptively transferred CD4 SP thymocytes using C57BL/6 mice as recipients. No clear differences in migration to secondary lymphoid organs were detected between cells from CD69 Tg and non-Tg mice 24 h after transfer (Fig. 6C). Interestingly, CD4 SP thymocytes from CD69 Tg mice did show a more rapid emigration from the blood to secondary lymphoid tissues (Fig. 6C). Taken together, these results indicate that constitutive CD69 surface expression does not influence T cell survival or result in abnormal compartmentalization of T cells within specific secondary lymphoid tissues.



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Fig. 6. Survival of CD4 SP thymocytes from CD69 Tg mice following adoptive transfer. CD4+ thymocytes from CD69 Tg (line #2028) mice and non-Tg littermates were purified by anti-CD8 and anti-B220 antibody depletion. Purified CD4+ T cells (3 x 106) without (A) or with (B and C) CFSE labeling were suspended in PBS and i.v. injected into non-irradiated Rag-2–/– (A and B) or C57BL/6 (C) mice. After 3 or 7 days, spleen cells and thymocytes of recipients were isolated, stained and analyzed by flow cytometry (A). Representative data of CFSE intensities and CD69 expression in CD4+ splenocytes from recipients 7 days after transfer are shown (B). Numbers of CFSE+ cells recovered from indicated organs after 24 h transfer are shown (C).

 
Thymus architecture in CD69 Tg mice
Examination of H & E-stained sections of thymus from CD69 Tg mice revealed an enlarged medulla relative to non-Tg mice but normal cortical and medullary demarcation (Fig. 7A). Localization of the medulla was confirmed by staining thymus sections for thymic medullary epithelial antigen (data not shown). We also examined the intrathymic localization of DP and SP thymocytes by confocal microscopy. As in non-Tg mice, most CD4 SP (green) and CD8 SP (red) cells were located in the medulla, whereas DP (yellow) cells localized to the cortex (Fig. 7B). Consistent with the results obtained by H & E staining, higher cell densities were observed in the medulla of CD69 Tg thymi and these cells consisted almost entirely of SP thymocytes. CD69 Tg mice contained slightly increased numbers of apoptotic cells in the medulla (data not shown). In addition, 3–4% of ex vivo SP thymocytes were Annexin V+ in CD69 Tg, compared to <1% in non-Tg mice (data not shown).



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Fig. 7. Immunohistochemical analysis of thymi from CD69 Tg. Thymi from 6- to 8-week-old CD69 Tg mice (right panel) and non-Tg littermates (left panel) were harvested and frozen for staining. H & E staining was performed to examine thymic architecture (A, x4 magnification). CD4 and CD8 staining was performed to localize thymocyte subsets and was analyzed by confocal microscopy (B, x40 magnification). Green, CD4; red, CD8.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD69 is widely used as marker for lymphocyte activation (14,15,17,1921). However, very little is known about its function in activated cells or its role in lymphocyte development, owing in part to its transient expression pattern. To explore the role of CD69 in T cell development, we generated Tg mice that constitutively express CD69 on developing T cells. The CD69 coding sequence was placed under the control of the human CD2 promotor and enhancer which directs T cell-specific, copy-number-dependent expression of transgenes in all thymocyte subsets and mature T cells (23). CD69 Tg mice displayed a transgene dose-dependent accumulation of mature SP thymocytes in the thymus and a concomitant reduction in the number of peripheral CD4 SP, CD8 SP and {gamma}{delta} T cells. Our results indicate that this phenotype is due to the failure of fully mature thymocytes to emigrate from the thymus. CD4 SP and CD8 SP thymocytes are produced with normal kinetics in CD69 Tg mice, and most of the SP cells that accumulate in the thymus are phenotypically and functionally mature. The accumulation of SP thymocytes is not due to re-entry of exported cells, since adoptively transferred SP thymocytes that express high surface levels of CD69 do not preferentially home to the thymus. Moreover, the paucity of peripheral T cells in CD69 Tg mice cannot be attributed to rapid death of exported cells or to the failure of cells to migrate to and seed secondary lymphoid organs. Finally, constitutive expression of CD69 results in expansion of the medullary region of the thymus, presumably due to the accumulation of SP thymocytes, but does not cause alterations in thymic architecture or effect the normal pattern of thymocyte localization within the thymus.

Perhaps the simplest interpretation of the CD69 Tg phenotype is that overexpression of CD69 prevents emigration of SP thymocytes by binding to its ligand and trapping cells in the medulla. Although our data do not exclude this interpretation, several lines of evidence favor a more complex mechanism. First, although a number of other cell surface molecules have been overexpressed in thymocytes, none of these Tg mice exhibit a phenotype similar to that observed in CD69 Tg mice, including mice that overexpress another C-type lectin-like receptor, Ly49A (32). Second, we were unable to induce the release of SP thymocytes from the thymus of newborn CD69 Tg mice by daily injection of antibody (anti-CD69) or CD69 tetramer (data not shown). Thus, blocking the interaction of CD69 with its putative ligand is not sufficient to reverse the effects of CD69 overexpression. These data raise the possibility that CD69 transmits a signal to mature thymocytes that inhibits their export from the thymus.

Cross-linking of CD69 on activated T cells or transfected mature cell lines elicits intracellular signals (e.g. Ca2+ influx and Erk activation), suggesting that it can function alone or in concert with the TCR to transduce physiologically relevant signals (33,34). However, we were unable to discern a difference in the Ca2+ or Erk activation responses in thymocytes from CD69 Tg and non-Tg mice following CD69 + TCR co-ligation and we did not observe a signaling response in thymocytes from CD69 Tg mice upon CD69 cross-linking (data not shown). In addition, thymocyte selection appeared unaffected in TCR Tg x CD69 Tg mice, indicating that the TCR signaling response was not influenced by CD69, at least during the period when thymocytes undergo selection in the thymus. Thus, these findings indicate that overexpression of CD69 does not impact signaling pathways downstream of the TCR in thymocytes.

The phenotype of CD69 Tg mice closely resembles that of pertussis toxin (PT) Tg mice (7). In PT Tg mice, thymocyte emigration is blocked, presumably because PT inhibits signaling through Gi-protein-linked chemokine receptors that regulate thymocyte export. As in CD69 Tg mice, functionally mature SP thymocytes accumulate in the thymus of PT Tg mice (7). More recently, it was shown that i.p. injection of PT inhibits the migration of SP thymocytes across the corticomedullary junction into the medulla (6). Interestingly, CD69 associates with a 40-kDa GTP binding (G{alpha} subunit) protein that is inhibited by PT (35). However, unlike mice injected with PT, SP thymocytes are restricted to the medulla in CD69 Tg mice and do not accumulate in the cortex. We also failed to note an obvious migration defect of adoptively transferred SP thymocytes to secondary lymphoid tissues in CD69 Tg mice. Interestingly, whereas SP thymocytes from PT Tg mice fail to migrate from blood into secondary lymphoid organs, SP thymocytes from CD69 Tg mice exhibit an accelerated egress from blood (Fig. 6C). This could reflect an enrichment in mature cells in the SP thymocyte populations from CD69 Tg mice relative to non-Tg mice. Alternatively, CD69 may function to augment chemotaxis initiated by certain chemokine receptors. Since signaling through chemokine receptors can also augment cell adhesion by activating integrins (36,37), this could potentially explain the failure of SP thymocytes to be exported from the thymus.

Notably, no alteration in T cell development has been observed in CD69–/– mice (22). However these results are not necessarily inconsistent with our data. For example, the failure to observe an effect on thymocyte emigration in CD69–/– mice could be explained by the compensatory activity of other molecule(s). In addition, the predicted phenotype of CD69–/– mice would be accelerated export of SP thymocytes, and this phenotype may not be obvious in adult mice since the mechanisms that regulate peripheral T cell numbers and T cell homeostasis may provide feedback that limits thymocyte emigration in T-replete hosts. Detailed analysis of the kinetics of thymocyte maturation and the phenotype of SP thymocytes and lymph node T cells should help to more accurately define the role of CD69 in T cell development.


    Acknowledgements
 
We would like to thank Dr Shoji Uehara, Dr Sandy Hayes and Dr Jeffrey R. Dorfman for helpful discussion and comments. We would like to thank Owen Schwartz for assistance in the confocal facility. K. J. W. is a Howard Hughes Medical Institute–National Institutes of Health Research Scholar.


    Abbreviation
 
APC—antigen-presenting cell

BrdU—5-bromodeoxyuridine

DN—double negative

DP—double positive

PFA—paraformaldehyde

PT—pertussis toxin

RTE—recent thymic emigrant

SP—single positive

Tg—transgenic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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