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The Journal of Immunology, 2004, 172: 7282-7288.
Copyright © 2004 by The American Association of Immunologists

Murine CD8+ Recent Thymic Emigrants are {alpha}E Integrin-Positive and CC Chemokine Ligand 25 Responsive1

Tracy L. Staton2, Brent Johnston, Eugene C. Butcher and Daniel J. Campbell

Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305; and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent thymic emigrants (RTE) are an important subpopulation of naive CD8+ T cells because of their ability to reconstitute a diverse immune system after periods of T cell depletion. In neonatal mice, the majority of peripheral T lymphocytes are RTE, cells that have recently left the thymus to populate the periphery. Postulating that these cells could have unique trafficking mechanisms, we compared adhesion molecule and chemokine receptor expression of neonatal RTE with mature adult lymphocytes. Neonatal CD8+ splenocytes uniformly express {alpha}E integrin and exhibit a high responsiveness to CC chemokine ligand (CCL25) (as compared with adult CD8+ splenocytes). Mature CD8+ thymocytes have a similar {alpha}E integrin+ CCL25 responsive phenotype, as do adult CD8+ RTE identified by intrathymic FITC injection. With increasing age, the frequency of CD8+ {alpha}E integrin+ splenocytes decreases, roughly correlating with thymic involution. Moreover, halting thymic output by thymectomy accelerates the age-dependent decline in peripheral CD8+ {alpha}E integrin+ RTE phenotype cells. Low expression of CD44 distinguishes these CD8+ RTE from a population of memory phenotype {alpha}E integrin+ CD8+ cells that are CD44high. We conclude that CD8+ RTE have unique adhesive and chemotactic properties that distinguish them from naive CD8+ T cells. These properties may enable specialized microenvironmental and cell-cell interactions contributing to the fate of RTE in the periphery during the early post-thymic period. This phenotype will also facilitate the identification and isolation of RTE for further studies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell development depends on molecular cues that guide cells through thymic microenvironments in which specialized epithelial and dendritic cells regulate positive and negative selection. Chemokines, chemokine receptors, and adhesion molecules are thought to direct these migration events (1, 2, 3). Once thymocytes complete their maturation into mature CD4+ or CD8+, MHC-restricted, and self-tolerant T cells, they leave the thymus and enter the periphery. Peripheral T cells that have recently left the thymus are termed recent thymic emigrants (RTE).3

Clinically, after periods of T cell depletion that can result from HIV infection, bone marrow transplantation, and chemotherapy, RTE play an important role in reconstituting diversity of the impaired peripheral T cell repertoire (4). In addition, RTE have a significant effect on the homeostatic regulation of the peripheral naive T cell pool. Although proper T cell numbers in the periphery are maintained by homeostatic proliferation, RTE play an important role by contributing new T cell specificities. It is believed that for a period of time following emigration, RTE have a "special status" in the periphery, with preferential access to survival factors and niches (5, 6). This allows T cells with new TCR specificities the opportunity to expand, survey the periphery, and interact with potential targets. Specific homing or adhesive properties may contribute to preferential RTE access to favorable environments.

The study of RTE has been hampered by a lack of cell surface markers to identify these cells. Monitoring TCR excision circle (TREC) levels by PCR has been one of the principle methods used to identify RTE (7), but this method cannot be used for analysis at the single cell level or for the isolation of viable RTE. TREC analysis describes the proliferative history of a population of cells and does not provide a direct measure of time since emigration, as processes such as cell division and cell death affect relative TREC levels irrespective of time in the periphery (8). TRECs are enriched in {alpha}E integrin+ CD8+ T cells in human blood (9), and T cell thymocyte Ag 1+ cells in chickens (10), but TREC analysis has not yet identified such phenotypes in mice. To more comprehensively study RTE at the single cell level, a specific surface phenotype for these cells must be identified.

We investigated the expression of selected adhesion molecules and chemokine receptors by CD8+ RTE in two settings in which RTE are readily identifiable. We examined splenocytes from neonatal mice, in which all peripheral T cells are RTE, as well as identified peripheral RTE in adult mice by intrathymic FITC labeling. We found that both neonatal and adult CD8+ RTE were {alpha}E integrin+ and responded to CC chemokine ligand (CCL)25, a phenotype that was shared with mature CD8 single positive (SP) cells in the thymus. Moreover, the frequency of {alpha}E integrin expression and the ability to respond to CCL25 decreased with age, and this decline was accelerated after thymectomy. Thus, unlike the bulk of naive CD8+ T cells, adult and neonatal CD8+ RTE are {alpha}E integrin+ and CCL25 responsive. This unique adhesion and chemotactic phenotype distinguishes RTE from most other naive CD8+ T cells and may play a role in the preferential survival or continuing functional maturation of RTE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c and C57BL6 mice were bred and housed at the Veterans Affairs Hospital (Palo Alto, CA).

Flow cytometry

The following directly conjugated mAbs from BD PharMingen (San Jose, CA) were used: anti-CD103/{alpha}E integrin (M290); anti-CD4 (RM4-5); anti-CD8{alpha} (53-6.7); anti-TCR{beta} (H57-597); anti-CD44 (1M7); anti-CD49d (SG31); anti-CD49e (5H10-27); anti-{beta}7 integrin (M293); anti-CD11a (2D7); anti-CD18 (C71/16); anti-CD62L (MEL-14); anti-CD29 (Ha2/5). Cells were stained in HBSS with 2% bovine serum and run on a FACSCalibur (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest software (BD Biosciences).

Chemotaxis assays

Migration assays were conducted as described (11). Briefly, 1 x 106 cells were added to the upper wells of 5-µm pore, polycarbonate 24-well tissue culture inserts (Costar, Cambridge, MA) in 100 µl, with 600 µl of diluted chemokine or medium in the bottom well. Murine chemokines were used at the following optimal concentrations: 250 nM CCL25, 100 µM CCL21, 50 nM CXCL12, 100 nM CXCL10, 100 nM CCL17, 100 nM CCL27, 250 nM CCL28, and 300 nM CXCL13 (R&D Systems, Minneapolis, MN). Three replicate wells were used for each chemokine and medium control. Cells were migrated in RPMI 1640 with 10% bovine serum at 37°C in 8% CO2 for 90 min. A 100-µl aliquot of migrated cells recovered from each well was counted by flow cytometry using beads as an internal standard. The remainder of the migrated cells was stained with directly conjugated mAbs. Percentage of migration was calculated for each flow cytometry-defined subset, by comparing its frequency in the input and migrated cell populations.

Intrathymic FITC injection

Intrathymic FITC injections were performed as described (12). Briefly, a ventral midline incision was made one-third down the sternum to expose the thymus. A Hamilton syringe was used to inject 10 µl of a 1 mg/ml FITC solution (Sigma-Aldrich, St. Louis, MO). The skin incision was closed with silk sutures and mice were allowed to recover under a heat source. At day 1 postinjection, cells in the blood were monitored for FITC incorporation. At day 4 after injection, blood, spleen, and lymph nodes were harvested for staining and chemotaxis.

RT-PCR

Splenocytes from mice injected intrathymically with FITC 4 days prior were sorted using a FACSVantage into FITC+ CD8+ and FITC+ CD8– subsets. Purity of cells was confirmed by reanalysis on a FACSCalibur. RNA was made from these cells using an RNAeasy kit (Qiagen, Valencia, CA). Total RNA was added to a reaction mixture containing cDNA synthesis buffer, dNTP, oligo(dT), RNase inhibitor, and Thermoscript RT to make cDNA (Invitrogen, Carlsbad, CA). cDNA was mixed with Taq polymerase, 10x reaction buffer, dNTP, and 5' and 3' primers designed to amplify a 500 bp product within the CCR9, CXCR4, and GAPDH genes (Qiagen). Reactions were run for 35 cycles.

Thymectomy

Mice thymectomized and sham-thymectomized at 4 wk were ordered from Taconic Farms (Germantown, NY) or were thymectomized in-house (13). Mice were tail bled at various days post-thymectomy and stained with directly conjugated mAbs. Complete thymectomy of each mouse was verified upon termination of experiment. Data were graphed using Prism software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal RTE express {alpha}E integrin

First, we analyzed the adult thymus for a phenotype that would distinguish CD8+ thymocytes in their latest stages of development from bulk peripheral T cells. In contrast to other thymocyte subsets, the majority of mature CD8+ SP thymocytes expressed {alpha}E integrin. A lower percentage of CD8+CD4low SP thymocytes, which represent an earlier less developmentally mature population, expressed {alpha}E integrin (Fig. 1B). The small percentage of double positive (DP) thymocytes that were {alpha}E integrin-positive were also TCRhigh, indicating that {alpha}E integrin was up-regulated after positive selection. All {alpha}E integrin+ CD8+ SP thymocytes were TCR{beta}+ (Fig. 1C). These CD8+CD4– ({alpha}E integrin+) SP cells represented mature thymocytes that are ready to exit the thymus, thus we reasoned that they may be most similar to RTE.



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  		FIGURE 1. CD8+ SP thymocytes express {alpha}E integrin and are TCR{beta}+. A, Adult thymocytes were stained with CD4 and CD8 and gated to delineate the DP and SP populations, as shown in B. B, Expression of {alpha}E integrin was determined on each population. C, Expression of {alpha}E integrin and TCR{beta} on gated CD8 SP cells. Data shown are from one mouse and are representative of more than nine mice from three independent experiments.

 
In mice, mature T cells do not exit the thymus and populate the periphery until after birth. At birth, the thymus contained 1–2% CD4 SP thymocytes but fewer than 1% CD8 SP thymocytes (Fig. 2A). By neonatal day 5, CD8 SP thymocytes began to appear and some mature TCR{alpha}{beta}+ CD8+ T cells had emigrated to the periphery. One or two weeks after birth, the thymus was exporting mature thymocytes into the periphery at increasing numbers (Fig. 2B). Thus, at neonatal day 5, all peripheral T cells had recently emigrated from the thymus and, by definition, were RTE. Neonatal RTE had similar V{beta} usage to adults, so early peripheral T cells did not represent the preferential export or expansion of a small number of V{beta}-restricted subsets of thymocytes (Fig. 3).



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  		FIGURE 2. Expression of the {alpha}E integrin on splenocytes declines with age. A, Thymocytes and splenocytes (B) from mice of various ages were stained with CD4 and CD8 to examine the kinetics of T cell development and emigration to the periphery as mice age. C, {alpha}E integrin expression by gated CD8+ splenocytes. Each plot is from one mouse and is representative of at least three individual experiments. D, Expression of {alpha}E integrin on CD8+ splenocytes was examined in mice of various ages. The percentage of CD8+ splenocytes that are {alpha}E integrin+ is plotted vs age. Each data point represents a single mouse. An exponential trendline is shown (R2 = 0.857).

 


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  		FIGURE 3. Neonatal CD8+ RTE have a diverse V{beta} repertoire, similar to adult CD8+ cells. Splenocytes from neonatal and adult mice were stained with a V{beta} panel, and V{beta} expression of neonatal CD8+ and adult CD8+ {alpha}E integrin+/– subsets were quantified. Shown is the percentage of each subset that was positive for that particular V{beta}. Data shown are the average from three mice in one experiment (with SD) and are representative of nine mice examined from three independent experiments.

 
Based on the fact that {alpha}E integrin was expressed on mature CD8 SP thymocytes, we examined {alpha}E integrin expression on CD8+ RTE in the neonate. In contrast to adult splenocytes, {alpha}E integrin was expressed by nearly all neonatal CD8+ splenocytes, with the frequency of cells of this phenotype declining with age (Fig. 2C). In mice, thymic output reaches a peak at adolescence and begins to decline around 3 mo of age (14). We observed that the pattern of {alpha}E integrin expression roughly paralleled thymic output (Fig. 2D). With increasing age there was a decrease in the percentage of splenic CD8+ T cells that were {alpha}E integrin+, reflecting a decrease in the proportion of RTE to naive cells in the splenic T cell pool. In support of the idea that RTE down-regulate {alpha}E integrin expression over time, a subset of sorted {alpha}E integrin+ splenocytes down-regulated their expression of {alpha}E integrin over a period of weeks when transferred into Thy1 mismatched recipients (data not shown).

Neonatal CD8+ RTE migrate to CCL25

Recent reports have shown that, unlike CD4 SP thymocytes, CD8 SP thymocytes preferentially maintain responsiveness to the thymic chemokine CCL25 (3, 15). Therefore we hypothesized that, similar to {alpha}E integrin expression, CCL25 responsiveness may distinguish RTE from bulk peripheral cells. A panel of eight chemokines (encompassing the ligands for most chemokine receptors expressed by T cells) was tested for differences in ability to attract neonatal and adult CD8+ splenocytes. Consistent with previous reports, neonatal CD8+ splenocytes migrated well to CCL25, whereas adult CD8+ splenocytes and CD4+ splenocytes migrated weakly in response to this chemokine (16). Also, neonatal CD4+ and CD8+ splenocytes migrated poorly to CXCL10 as compared with their adult counterparts, consistent with neonatal T cells being predominantly naive (Fig. 4A).



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  		FIGURE 4. Neonatal {alpha}E integrin+ CD8+ splenocytes migrate to CCL25. A, Neonatal and adult CD4+ and CD8+ splenocytes were migrated to a panel of chemokines (representing the ligands for most chemokine receptors). *, Undetectable migration. B, Adult thymocytes and splenocytes were migrated to CCL25 and the percentage of migration for the indicated subsets was determined. C, Neonatal and adult splenocytes were migrated to CCL25 and stained with {alpha}E integrin to determine subset specific migration. In all plots, percentage of migration is shown with background migration subtracted. Error bars represent SD of triplicate wells. Data are representative of at least three independent experiments.

 
Because CCL25 is involved in thymocyte migration, we characterized the changes in thymocyte responsiveness to CCL25 and compared this with the response of RTE and of bulk naive T cells in the periphery, focusing specifically on the CD8+ compartment. We found that DP thymocytes migrated to CCL25, and CD8 SP thymocytes showed an enhanced CCL25 responsiveness as compared with CD4 SP thymocytes. In the spleen, responsiveness to CCL25 was maintained on {alpha}E integrin+, but not {alpha}E integrin–, CD8+ cells, whereas neither population of CD4+ cells migrated to CCL25 (Fig. 4, A and C). In the neonatal spleen, CD8+ T cells responded to CCL25, suggesting that neonatal CD8+ RTE retained responsiveness to CCL25 upon emigration to the periphery (Fig. 4C). In contrast, the {alpha}E integrin– CD8+ adult splenocytes (which constituted the majority of adult CD8+ T cells) migrated poorly to CCL25, whereas {alpha}E integrin+ CD8+ adult splenocytes responded to CCL25 (Fig. 4C). Therefore, the CCL25-responsive RTE population of peripheral CD8+ T cells corresponded to the CD8+ {alpha}E integrin+ population, in both the adult and the neonate. This correlated with data showing CCR9 (the receptor for CCL25) expression on some naive CD8+ T cells from secondary lymphoid organs (16, 17, 18). The CCL25 response was specific to the CD8+ compartment, as CD4+ {alpha}E integrin+ splenocytes did not migrate to CCL25 (Fig. 4B).

Adult CD8+ RTE are {alpha}E integrin+ and CCL25 responsive

The data presented so far have described a unique phenotype of CD8+ RTE in neonatal mice. We next examined emigrants in adult mice, using intrathymic FITC injection to identify adult RTE. A fluorescent dye, FITC, injected into the thymus is taken up by developing thymocytes, and retained after emigration to the periphery so that all peripheral FITC+ cells are RTE (12, 14). Four days after intrathymic FITC injection, only TCR{beta}+ cells were FITC+, indicating that intrathymic injection delivered FITC specifically to thymus (Fig. 5A). FITC+ CD8+ RTE from spleen and peripheral blood were analyzed 1–6 days after intrathymic injection and found to be uniformly {alpha}E integrin+, with a representative time point shown in Fig. 5B. Expression of a panel of seven additional T cell-associated integrins was examined on FITC+ CD8+ RTE, FITC– {alpha}E integrin+ CD8+ cells, and FITC– {alpha}E integrin– CD8+ cells from spleen. These included CD11a, CD11b, {alpha}4, {alpha}5, {beta}1, {beta}2, and {beta}7. Expression of all integrins was similar, except for {beta}7 integrin (the {beta}-chain that pairs with {alpha}E integrin), which was expressed highest on FITC+ CD8+ cells, intermediate on FITC– {alpha}E integrin+ CD8+ cells, but low on FITC– {alpha}E integrin–CD8+ cells (data not shown).



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  		FIGURE 5. Adult RTE express {alpha}E integrin and migrate to CCL25. Adult mice were injected intrathymically with FITC to identify adult RTE. Mice were analyzed 4 days postinjection. A, PBMCs were stained with TCR{beta} and CD19 to confirm that FITC only labeled thymocytes and did not label all cells in the blood. B, FITC+ and FITC– splenocytes were gated and analyzed for expression of {alpha}E integrin on CD8+ cells. C, Migration of FITC+ splenocytes and bulk CD8+ splenocytes to CCL25 were compared. The percentage of migration for each subset is shown with background migration subtracted. Error bars represent SD of triplicate wells. D, FITC+ CD8+ and FITC+ CD8– cells were sorted using a FACSVantage cell sorter. RNA was made from each of these populations and expression of CCR9, CXCR4, and GAPDH was determined by RT-PCR. All data are representative of at least three independent experiments.

 
As was the case with neonatal CD8+ RTE, FITC+ CD8+ adult RTE also migrated to CCL25 (Fig. 5C). Many FITC– cells were also {alpha}E integrin+ and responded to CCL25, consistent with the fact that intrathymic FITC injection labels only a subset of thymocytes near the injection site. Taken together, these data demonstrate that {alpha}E integrin expression and CCL25 responsiveness are characteristic of both adult and neonatal CD8+ RTE. However, it is interesting to note that neonatal CD8+ RTE consistently migrated two-fold better to CCL25 than adult CD8+ RTE (compare Figs. 4C and 5B). The significance of this enhanced migration is unknown.

To confirm CCR9 expression on CD8+ RTE, both FITC+ CD8+ and FITC+ CD8– T cell subsets were sorted from spleen 4 days after intrathymic FITC injection. RNA was isolated and RT-PCR performed, using CCR9 and CXCR4 specific primers. Only the FITC+ CD8+ subset expressed high levels of CCR9, whereas both expressed CXCR4 (Fig. 5D). These PCR results correlate with the chemotaxis results shown in Fig. 4A.

The experiments described were conducted in the BALB/c strain. Similar experiments were also performed in another strain, C57BL6, in which the CD8+ RTE phenotype is conserved in neonates and adults (data not shown). Intrathymic FITC injections in C57BL6 mice confirmed that adult CD8+ RTE were {alpha}E integrin+, and CD8+ {alpha}E integrin+ cells migrated to CCL25 at similar levels in both strains. However, the relative frequency of {alpha}E integrin+ CD8+ cells in C57BL6 mice is always higher than in age-matched BALB/c animals. This may reflect a more prolonged expression of {alpha}E integrin by CD8+ T cells after emigrating from the thymus in this strain.

Adult thymectomized mice progressively lose {alpha}E integrin+ CD8+ RTE in the periphery

To examine the role of the thymus in the production and maintenance of the peripheral CD8+ {alpha}E integrin+ T cell pool, we examined the effect of thymectomy on the frequency of peripheral {alpha}E integrin+ CD8+ T cells. Mice were thymectomized at 4 wk of age and the percentage of CD8+ {alpha}E integrin+ cells was monitored in blood and lymphoid tissues for over 3 mo. We observed that the percentage of CD8+ {alpha}E integrin+ cells in the blood declined faster in thymectomized mice than in nonthymectomized mice during the first 3 wk post-thymectomy, presumably reflecting the lack of thymic output (Fig. 6). Mock-thymectomized mice were similar to nonthymectomized mice (data not shown). Overall, the percentage of CD8+ {alpha}E integrin+ cells declined over time in both thymectomized and nonthymectomized mice because the rate of thymic export decreases with age, as shown in Fig. 2. However, the rapid disappearance of {alpha}E integrin+ CD8+ cells after thymectomy supported the idea that these cells were in fact RTE. There was no difference in the percentage of the small subset of CD4+ {alpha}E integrin+ cells between thymectomized and nonthymectomized mice (data not shown). These experiments indicate that the percentage of {alpha}E integrin+ CD8+ T cells can be used to detect changes in thymic output of adult mice.



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  		FIGURE 6. Percentage of {alpha}E integrin+ CD8+ cells declines in thymectomized mice over time, with remaining {alpha}E integrin+ CD8+ cells being of a memory phenotype. A, Mice were thymectomized at 4 wk of age. Blood was taken various days after thymectomy to monitor the percentage of {alpha}E integrin+ CD8+ cells in the blood. Each point on the graph is the percentage of CD8+ cells that are {alpha}E integrin+ and represents an average of more than ten mice. The day 0 value was extrapolated from the data points fitted to this line (this value is similar to previous observations at this age, as in Fig. 2). Data from the nonthymectomized mice were best fit by linear regression (R2 = 0.98). Data from thymectomized mice were best fit by nonlinear regression to an exponential decay curve (R2 = 0.99). B, CD44 staining of RTE vs {alpha}E integrin+ non-RTE in adult mice. To identify adult RTE, mice were injected intrathymically with FITC. FITC+ splenocytes were analyzed 4 days postinjection. To evaluate adult non-RTE, mice were thymectomized at 5 wk of age and analyzed 4 mo later. CD44 was measured on {alpha}E integrin+ CD8+ cells from both groups and is shown as a histogram. Shaded histogram represents FITC+ CD8+ RTE, solid histogram represents {alpha}E integrin+ CD8+ cells from thymectomized mice. Data are representative of three independent experiments.

 
Although the percentage of {alpha}E integrin+ CD8+ cells declined after thymectomy, 6 wk post-thymectomy a plateau value of 10–15% {alpha}E integrin+ CD8+ cells was reached (Fig. 6A). The percentage of {alpha}E integrin+ CD8+ cells in the blood remained at 10–15% as far out as 6 mo post-thymectomy (data not shown). A similar phenomenon occurred as mice age, in which 10–15% of total CD8+ cells remained {alpha}E integrin+ in mice older than 3 mo (Fig. 2). {alpha}E Integrin was originally identified as a marker of mucosal intraepithelial memory T cells (19, 20, 21) and since then has been found to be expressed on a variety of other tissue-specific memory cells (22, 23, 24). Thus, it was likely that these {alpha}E integrin+ CD8+ cells were memory cells that recirculated to the blood and spleen, and would be expected to have a memory phenotype that distinguishes them from the naive {alpha}E integrin+ CD8+ RTE. Therefore, we examined expression of the defined memory marker CD44 by cells from mice injected intrathymically with FITC (to obtain a pure RTE population) and cells from mice 4 mo post-thymectomy (to obtain a pure non-RTE population) (Fig. 6B). The FITC+ CD8+ RTE population was CD44low, and clearly differed from the CD44high {alpha}E integrin+ CD8+ cells from thymectomized mice. Thus, the CD8+ RTE phenotype is more strictly defined as {alpha}E integrin+ CD44low, and CCL25 responsive.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RTE play an important role in the immune system, as they link the thymus to the peripheral naive T cell pool and replenish its diversity. Taking advantage of the unique patterns of expression of adhesion and trafficking molecules that characterize functionally and developmentally specialized lymphocyte subsets, we sought to identify and characterize features unique to murine RTE. We present a murine CD8+ RTE phenotype that will facilitate isolation and further study of these cells, and may be useful for monitoring thymic output.

We showed that {alpha}E integrin was expressed specifically on mature CD8+ thymocytes, and that the majority of neonatal but not adult CD8+ splenocytes expressed {alpha}E integrin. We next examined chemokine responsiveness in the thymus and spleen of neonates and adults, and found that neonatal CD8+ T cells selectively displayed high responsiveness to CCL25. Only the {alpha}E integrin+ cells in the neonate and adult migrated to CCL25, suggesting a specific functionality to this integrin-chemokine receptor pairing. These data present a specific murine CD8+ RTE phenotype in neonates and adults, distinguished from the phenotype of the majority of adult naive phenotype CD8 cells. Consistent with the notion that CD8+ RTE are {alpha}E integrin+, a progressive decrease in the frequency of these cells is observed with age and decreasing thymic output, and this is accelerated by thymectomy. In older mice and thymectomized mice, there is a subset of CD8+ cells that remain {alpha}E integrin+. However, these residual {alpha}E integrin+ CD8+ cells can be distinguished from RTE by their high level expression of CD44.

As of yet, it is unclear why RTE express {alpha}E integrin and CCR9 and what roles these molecules play in the thymus and periphery. There is evidence that RTE may have a special status in the periphery for a defined period of time (~3 wk), during which RTE are given priority for niches, comprised of survival factors including IL-7 and MHC-self peptide complexes on APC (6). If after ~3 wk these cells do not receive the proper stimulus they lose priority and become more susceptible to displacement from niches by fresh RTE, contributing to repertoire turnover throughout life (5). We speculate that during this period, the unique chemotactic and adhesive phenotype of RTE may give them preferential access to physical compartments, allowing them to outcompete older naive CD8 T cells.

Data from {alpha}E integrin–/– mice suggest that expression of {alpha}E integrin allows CD8+{alpha}{beta}+ T cells to access or be retained in the skin epidermis and mucosal epithelium (25, 26, 27). Similarly, CCR9 is thought to be involved in providing access for T cells to the intestinal epithelium and lamina propria of the small intestine (28). Accordingly, {alpha}E integrin expression on memory/effector CD8+ T cells is restricted to certain mucosal or cutaneous populations, whereas CCR9 is found predominantly on small intestinal T cells (29). It has long been hypothesized that T cells can be tolerized to peripheral self-Ags upon leaving the thymus during the neonatal period, perhaps by trafficking through peripheral tissues in which they encounter Ag in the absence of necessary costimulatory signals (30, 31). Thus, {alpha}E integrin and CCR9 may direct early trafficking of RTE to peripheral sites, and this may constitute a final educational step in T cell maturation outside the thymus. This effect would be most pronounced the neonatal period because in neonatal mice all T cells are RTE. Indeed, neonatal RTE, identified by intrathymic FITC injection, have been reported in sections of the small intestine (32).

A further possibility is that {alpha}E integrin and CCR9 function predominantly in thymocyte development, rather than in the periphery. Selective expression of {alpha}E integrin and retention of CCR9 on CD8 SP thymocytes is consistent with a specific role for these molecules in CD8 T cell development. Thymus colonization is delayed in CCR9–/– mice (33) and CCR9 is up-regulated after pre-TCR signaling (15, 28), suggesting an important role for CCR9 mediated signals in early thymocyte development. CCR9 and {alpha}E integrin may also be involved in targeted migration and cell-cell interactions important for cell survival and positioning during/after positive selection. For example, {alpha}E integrin-E cadherin interactions between thymocytes and thymic epithelial cells have been shown to induce proliferation of thymocytes (34). Also, CCL25 is expressed on both cortical and medullary epithelium in the thymus, possibly directing migrations of less mature cortical thymocytes as well as more mature medullary CD8 SP cells (35). The expression of {alpha}E integrin and CCR9 by RTE may therefore reflect these molecules’ importance in CD8+ T cell development. However, a specific defect in CD8+ T cell development has not yet been observed in CCR9–/– or {alpha}E integrin–/– mice.

In conclusion, we have shown that murine CD8+ RTE express {alpha}E integrin and respond to CCL25, which distinguishes them from the bulk pool of naive CD8+ T cells. As markers, {alpha}E integrin and CCR9 will aid in studies of the unique properties of RTE, including the potential for selective homeostatic and/or trafficking properties that may be mediated by these molecules. These markers may also prove useful for studying thymic output, particularly during immune system reconstitution following irradiation, chemotherapy, or other lymphocyte depleting regimens.


   Acknowledgments
 
We thank Monte M. Winslow for critical review of the manuscript and thoughtful discussions and Henrik Thorlacius for helpful comments.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI47822, GM37734, and HL67674 (to E.C.B.), by an award from the Department of Veterans Affairs, and by the FACS Core Facility of the Stanford Digestive Disease Center under a National Institutes of Health Grant DK-56339 (to E.C.B.). Back

2 Address correspondence and reprint requests to Tracy L. Staton, Veterans Affairs Palo Alto, 3801 Miranda Avenue, 154B, Palo Alto, CA 94304. E-mail address: tstaton{at}stanford.edu Back

3 Abbreviations used in this paper: RTE, recent thymic emigrant; TREC, TCR excision circle; DP, double positive; SP, single positive. Back

Received for publication September 11, 2003. Accepted for publication April 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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