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The Journal of Immunology, 2005, 175: 2270-2277.
Copyright © 2005 by The American Association of Immunologists

Control of Recent Thymic Emigrant Survival by Positive Selection Signals and Early Growth Response Gene 11

Frederick J. Schnell and Gilbert J. Kersh2

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Early growth response gene 1 (Egr1) is a transcriptional regulator whose expression can be induced by multiple signals including the TCR. Egr1 has been shown to promote positive selection, but an investigation of its role in T cell homeostasis has not been reported. The possibility that similar signals control both positive selection and peripheral T cell homeostasis led us to investigate the role of Egr1 in the maintenance of peripheral T cells. We have found that on TCR transgenic backgrounds, Egr1-deficient mice have a reduction in their number of naive T cells. Although Egr1-deficient animals have a low percentage of mature thymocytes due to inefficient positive selection, the absolute number of mature thymocytes is only slightly reduced due to increased thymus size in Egr1-deficient mice. Despite possessing near normal numbers of mature thymocytes, we find that Egr1-deficient mice have poor accumulation of recent thymic emigrants (RTE) in the periphery. The poor accumulation of RTE in Egr1-deficient mice appears to originate from decreased survival of mature thymocytes and RTE, which we have observed both in vitro and in vivo. These findings suggest that an Egr1-mediated signal during positive selection promotes not only the production of single positive thymocytes, but also the survival of selected thymocytes until they can become established in the periphery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Early growth response gene 1 (Egr1)3 is a transcriptional regulator with a zinc-finger DNA binding domain that is quickly induced upon external stimulation in many different cell types (1, 2). Egr1 is expressed in thymocytes and peripheral T cells and its expression is rapidly induced upon TCR engagement in an ERK-dependent manner (3, 4). Egr1 was one of the first transcription factors found to play a role in translating ERK activation into changes in gene expression during positive selection (5). Egr1-deficient mice have inefficient positive selection, resulting in a reduced percentage of CD4 and CD8 single positive (SP) mature cells in the thymus. However, the absolute number of mature thymocytes is only slightly reduced in Egr1-deficient mice due to the fact that these thymuses are much larger than normal (5). Egr1 also appears to be involved in signaling events induced in T cells subsequent to interaction with unprimed self APCs. Our group recently discovered that Egr1 is induced in peripheral T cells after interaction with syngeneic APC in the absence of exogenous Ag (6).

The fact that Egr1 is required for efficient positive selection, and that Egr1 can be induced downstream of the TCR through interaction with self APC, raises the possibility that Egr1 is a regulator of peripheral T cell homeostasis. Evidence suggests that naive T cell homeostasis depends on signals generated from IL-7 and TCR stimulation (7, 8, 9, 10, 11, 12, 13). There is evidence that similar signals through the TCR are required for both positive selection in the thymus and the regulation of naive T cell survival in the periphery. It is known that peripheral T cells have a partially phosphorylated CD3 {zeta}-chain that is dependent on contact with MHC (9, 14). This fact suggests that naive T cells are constantly receiving weak signals from contact with APC. Another report has shown a dependence on the Src family kinases p56lck and p59fyn for long-term survival of naive T cells (9). When both lck and fyn expression were disrupted in peripheral T cells, naive T cell survival was compromised. This also suggests that a weak form of TCR signaling that activates lck and/or fyn is required for maintenance of peripheral T cells. Other reports have shown a dependence on surface TCR expression in the maintenance of naive T cell numbers. Naive T cells decayed at a faster rate when surface TCR{alpha} expression was ablated, again reaffirming the need for TCR signaling in the maintenance of naive T cell survival (11, 12). Homeostatic proliferation of naive T cells in response to lymphopenia requires TCR interaction with peptide/MHC ligands that are similar to the ligands involved in positive selection (15, 16, 17, 18, 19). Another signaling molecule downstream of the TCR that has been shown to play a role in preserving naive T cell homeostasis is RasGRP1. RasGRP1 is a Ras guanine nucleotide releasing protein exchange factor whose activity results in the specific activation of ERK (20). RasGRP1-deficient mice have reduced numbers of peripheral T cells because of a lack of T cell export and reduced peripheral expansion (13). Taken together, these reports support the idea that TCR expression and ERK activation are required for naive T cell survival and/or proliferation, just as they are required for positive selection.

Because RasGRP1-mediated ERK activation is required to maintain a full T cell compartment and Egr1 is activated in a TCR and ERK-dependent manner in naive T cells upon incubation with syngeneic APC, we sought to examine the role of Egr1 in maintaining T cell homeostasis. We have found that Egr1-deficient mice have reduced numbers of naive CD4 T cells in the periphery when bred onto TCR transgenic backgrounds. However, we also unexpectedly found that long-term survival and homeostatic proliferation of mature T cells was normal in the absence of Egr1. These results led us to more carefully examine the cells that undergo positive selection in Egr1-deficient mice. We have found that mature TCR transgenic thymocytes positively selected in the absence of Egr1 poorly survive both in vivo and in vitro. The increased apoptosis results in the establishment of only small numbers of Egr1-deficient recent thymic emigrants (RTE) in the periphery, thus leading to low numbers of naive T cells in Egr1-deficient mice. These findings suggest that the quality of signals generated during positive selection can have a lasting impact on the ability of thymocytes to survive even after exiting the thymus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Egr1-deficient mice were generated as described and backcrossed to the C57BL/6 background over seven generations (21). The targeted allele was then crossed two generations to B6.AKR and screened for the H-2k/k genotype to generate Egr1-deficient mice on the B6.AKR background. N3.L2 mice (3.L2 TCR transgenic mice) were generated as described and maintained on a B6.AKR background (22, 23). B6.PL mice were bred onto the B6.AKR background and were screened for the Thy1.1 genotype. B6.AKR and B6.PL mice were purchased from The Jackson Laboratory. OT-1 mice were kindly provided by Dr. J. Kapp (Emory University School of Medicine, Atlanta, GA). All procedures were approved by the Emory University Institutional Animal Care and Use Committee.

Flow cytometry

The following Abs used in this study were purchased from BD Pharmingen: CD44-biotin (IM7), CD4-PE (GK1.5), CD69-biotin (H1.2F3), and anti-BrdU-FITC. The following reagents were purchased from Caltag Laboratories: anti-CD8 TC (CD8a), CD62L-FITC, Annexin VFITC, and streptavidin-allophycocyanin. The Ab against the 3.L2 TCR clonotype (Cab) was prepared as described previously (24). Thymocyte apoptosis was analyzed using the Annexin VFITC apoptosis detection kit I (BD Pharmingen) according to the manufacturer’s instructions. For the in vitro survival assay, thymocytes (3 x 106 cells/well in a 24-well plate) were cultured for various periods of time and analyzed using the annexin V apoptosis detection kit.

BrdU labeling

BrdU incorporation was detected with the BrdU flow kit (BD Pharmingen). For long-term pulse, mice were injected i.p. with 1 mg of BrdU dissolved in PBS each day for 9 consecutive days. Mice were tail bled at indicated time points, and PBL were stained for surface markers. RBC were lysed with RBC Lysing buffer (Sigma-Aldrich) and then fixed in Cytofix/Cytoperm buffer, permeabilized in Cytoperm Plus buffer, and then reincubated in Cytofix/Cytoperm buffer. Cells were then treated with DNase to expose BrdU epitopes, and immunofluorescent staining was performed with anti-BrdU FITC and analyzed by FACSCalibur. For short-term pulse, 1 mg of BrdU was injected i.p., and mice were sacrificed 5 h later.

Adoptive transfer

For analysis of lymphopenia-induced proliferation, cells were labeled with the fluorescent dye CFSE (Molecular Probes). Briefly, 107 cells (from a single-cell suspension of splenic lymphocytes) were washed twice with room temperature PBS and incubated with 2.5 µM CFSE in a volume of 2 ml for 5 min. The reaction was quenched by the addition of 400 µl of FBS for 30 s, and the cells were immediately washed twice with PBS. Cells were then transferred i.v. into mice that had been sublethally irradiated (600 rad) 3–24 h earlier. Fourteen days after transfer, spleen and inguinal lymph node cells were analyzed for CFSE expression by flow cytometry. For N3.L2 thymocyte adoptive transfers, thymocytes were labeled with CFSE, and equal numbers of CD4SP thymocytes were injected i.v. into B6.AKR Thy-1.1 recipients. At 24 h after transfer, >95% of the transferred cells identified in inguinal lymph nodes were CD4 or CD8 SP cells.

Thymus cell export

Mice were anesthetized, the upper chest opened, and the thymus lobes exposed. Each thymus lobe was injected with 10 µl of FITC (1 mg/ml), which resulted in the labeling of 50–100% of all thymocytes. Mice were sacrificed 48 h later, and the RTE present in the spleen and inguinal lymph nodes were identified by flow cytometry as live FITC+ cells expressing CD4 or CD8. The number of emigrants was adjusted based on the percentage of labeling of thymocytes in the corresponding thymus.

T cell stimulation

Splenocytes were added to a 96-well plate (5 x 106 cells/well) and hemoglobin (Hb)(64–76) peptide was added at indicated concentrations. Cells were incubated at 37°C for 2 days and then pulsed with [3H]thymidine (0.5 µCi/well). The next day incorporation of [3H]thymidine was measured with a scintillation counter. The sequence of Hb(64–76) is GKKVITAFNEGLK. Peptides were synthesized by the Emory University Microchemical Facility (Atlanta, GA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCR transgenic Egr1-deficient mice have reduced numbers of naive T cells

Egr1-deficient CD4/CD8 double positive (DP) thymocytes are not able to fully transduce TCR-mediated positive selection signals resulting in a reduced percentage of mature thymocytes (5). Because some studies have shown that signals through the TCR are required for naive T cell survival in the periphery (9, 10, 11, 12, 13), we have investigated the possibility that Egr1 regulates peripheral T cell homeostasis downstream of TCR signals. Therefore, we have compared Egr1+/+ and Egr1–/– lymphoid organs on both a normal (B6.AKR), 3.L2 TCR transgenic (N3.L2), and OT-1 TCR transgenic background. The 3.L2 TCR is specific for a peptide derived from the d allele of murine Hb presented by I-Ek (24). The N3.L2 line is maintained on a B6.AKR background (H-2k and Hb s allele), and cells bearing the transgenic TCR are positively selected into the CD4 lineage. The OT-1 TCR is specific for OVA257–264 (SIINFEKL) complexed to H-2Kb, and the majority of T cells in these mice express the TCR {alpha} and {beta} transgenes and are selected into the CD8 lineage (25).

Comparison of peripheral lymphoid organs between Egr1+/+ and Egr1–/– mice on different TCR transgenic backgrounds revealed that the percentage of naive T cells bearing the 3.L2 and OT-1 TCR in Egr1–/– animals is very low (Fig. 1). In N3.L2 Egr1+/+ mice, 7.1% of CD44low splenocytes express CD4 and have high levels of the 3.L2 TCR, whereas only 0.9% of CD44low cells are 3.L2highCD4+ in N3.L2 Egr1–/– spleens (Fig. 1A). Examination of CD8 T cell numbers in OT-1 Egr1–/– mice revealed a similar phenotype. In OT-1 Egr1+/+ mice, 34.3% of CD44low cells express CD8, whereas only 16.7% of CD44low cells express CD8 in OT-1 Egr1–/– mice. If we examine absolute numbers of cells in the spleen, we find that N3.L2 Egr1+/+ mice have on average 2.8 times as many 3.L2highCD44lowCD4+ cells as N3.L2 Egr1–/– mice (Fig. 1B). However, this effect is dependent on the expression of a TCR transgene. Non-TCR transgenic Egr1–/– mice have normal numbers of naive peripheral T cells (Fig. 1C), and N3.L2 Egr1–/– mice have normal numbers of 3.L2low T cells (Fig. 1C). The 3.L2low T cells in N3.L2 mice have been shown to express endogenously rearranged TCR (data not shown). Thus, it appears that Egr1 is required for the maintenance of a full peripheral T cell pool when the TCR is fixed by the presence of a TCR transgene. We presume that in the absence of a TCR transgene, TCR specificities that support normal naive T cell numbers in an Egr1-independent manner can be selected in the thymus. Therefore, Egr1 contributes to the normal population of the peripheral lymphoid organs by naive T cells.



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  		FIGURE 1. TCR transgenic Egr1-deficient mice have reduced numbers of peripheral T cells. Cell suspensions were prepared from spleens of Egr1+/+ and Egr1–/– mice on either a N.3L2, OT-1, or non-TCR transgenic background and stained for CD4, CD8, CD44, and 3.L2 TCR. A, The dot plots display staining for CD4 and 3.L2 TCR after gating on CD44low cells. The percentage in the dot plots indicates the CD44low cells that are 3.L2highCD4+. B, The histograms show staining for CD8 after gating on CD44low cells. The percentage on the gates indicates the CD44low cells that are CD8+. C, Bar charts show total number of CD4 T cells in 3.L2 TCR transgenic (left) and non-TCR transgenic (non-TCRtg, right) mice on either an Egr1+/+ or Egr1–/– background. Data represents between six and eight mice per group ± SEM. Significance was determined by an unpaired two-tailed Student’s t test (*, p < 0.0001).

 
The loss of naive 3.L2highCD4 T cells in N3.L2 Egr1-deficient mice could be attributed to a reduced ability to proliferate, much like RasGRP1-deficient mice (13). We therefore investigated the ability of 3.L2high Egr1–/– T cells to respond to agonist peptide stimulation and to proliferate in lymphopenic conditions. We pulsed equal numbers of splenocytes from N3.L2 Egr1–/– and control mice with Hb peptide (Hb(64–76)) and measured proliferation 24–48 h later. As can be seen in Fig. 2A, N3.L2 Egr1-deficient T cells were able to fully respond to agonist peptide stimulation. It therefore does not appear that Egr1 is required to transmit strong peptide/MHC signals through the TCR. We next tested whether Egr1 is required for T cell proliferation in lymphopenic conditions. Equal numbers of CFSE-labeled CD4 T cells derived from N.3L2 Egr1+/+ and N3.L2 Egr1–/– mice were i.v. injected into sublethally irradiated hosts, and 14 days postinjection, lymphoid organs were harvested to examine the number of cycles of proliferation. As can be seen in Fig. 2B, 3.L2highCD4+ Egr1-deficient cells were able to undergo homeostatic proliferation as well as 3.L2highCD4+ control cells. These results demonstrate that Egr1 is not required for proliferation in response to agonist peptide or lymphopenic conditions. It is therefore improbable that the loss of naive, peripheral T cells in N3.L2 Egr1–/– mice is due to impaired proliferation.



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  		FIGURE 2. N3.L2 Egr1-deficient T cells have a normal response to agonist peptide stimulation and lymphopenia. A, N3.L2 Egr1+/+ and N3.L2 Egr1–/– splenocytes were cultured with various concentrations of Hb peptide (64–76) and proliferation was measured by incorporation of [3H]thymidine ± SEM. B, Splenocytes from N3.L2 Egr1+/+ (top) and N3.L2 Egr1–/– (bottom) mice were CFSE-labeled and injected into sublethally irradiated (600 rad) congenic mice and donor cells from host lymphoid organs were analyzed 14 days later. Shown are CFSE profiles of donor 3.L2highCD4+ cells. Data are representative of three to five experiments.

 
Reduced accumulation of RTE in the periphery of N3.L2 Egr1-deficient mice

We have also tested the possibility that the disappearance of 3.L2high T cells in N3.L2 Egr1-deficient mice is due to a decreased ability to survive. Numerous groups have shown that signals transmitted through the TCR are required for naive CD4 T cell survival (9, 10, 11, 12, 13). TCR mediated prosurvival signals could be inefficiently transmitted because of the lack of Egr1, thereby reducing the lifespan of Egr1-deficient T cells. We tested this possibility by doing a long-term pulse-chase experiment with BrdU to examine the survival of N3.L2 Egr1-deficient T cells. N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were pulsed with BrdU for 9 days and then chased for 55 days to examine the rate of decay of labeled cells. In the absence of cognate Ag, peripheral naive CD4 T cells proliferate at very low levels. Therefore, almost all of the labeled naive T cells that appear in the periphery have incorporated BrdU while proliferating in the thymus during the pulse period. As can be seen in Fig. 3A, N3.L2 Egr1+/+ mice have a higher percentage of labeled 3.L2high cells than N3.L2 Egr1–/– mice at both 10 and 20 days after the beginning of BrdU administration. The average percentage of labeling of 3.L2high cells in N3.L2 Egr1–/– mice is only 4.1% at day 10 with a peak of 9.1% at day 20. This is compared with an average percentage of labeling of 9.6% at day 10 and a peak of 16.2% at day 20 for N3.L2 Egr1+/+ littermate controls. However, from day 30 to day 65, N3.L2 Egr1–/– and N3.L2 Egr1+/+ mice maintain similar percentages of labeled cells (Fig. 3A). It therefore appears that the long-term survival of Egr1-deficient 3.L2high T cells is normal. We cannot rule out the possibility that Egr1-deficient T cells are able to compensate for increased apoptosis at later stages by proliferating more. However, this option seems unlikely due to the very low rates of proliferation that we observe in naive peripheral T cells in both Egr1+/+ and Egr1–/– mice. Consistent with our previous data, 3.L2low Egr1-deficient T cells appeared to accumulate in the periphery and decayed normally during the time course (Fig. 3A).



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  		FIGURE 3. N3.L2 Egr1-deficient mice have reduced numbers of RTE. A, N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were pulsed with BrdU for 9 days and then chased for 55 days. The data show the percentage of BrdU labeling by 3.L2high and 3.L2low subsets of PBL CD4+ cells from N3.L2 Egr1+/+ or N3.L2 Egr1–/– mice. Arrow denotes when pulse was terminated. Data represent an average of two mice per group per experiment and three independent experiments. B, Non-TCR transgenic (Non-TCRtg) Egr1+/+ (n = 4) and Egr1–/– (n = 4) and N3.L2 Egr1+/+ (n = 7) and N3.L2 Egr1–/– (n = 7) mice were anesthetized and 10 µg of FITC was directly injected into each thymic lobe. Spleens and inguinal lymph nodes were harvested 48 h later and examined for FITC+ RTE. Each Egr1–/– mouse was paired with an age-matched control and the data are presented as the number of CD4+ RTE in an Egr1–/– mouse divided by the number of RTE in a paired age-matched control ± SEM. The number of FITC+ cells recovered per spleen (adjusted for labeling efficiency) in three age-matched pairs was 1.6 x 104 3.L2high cells and 5.4 x 104 3.L2low cells in N.3L2 Egr1–/– mice, and 3.5 x 104 3.L2high cells and 6.0 x 104 3.L2low cells in N.3L2 Egr1+/+ mice. The difference between the number of 3.L2high RTE found in N3.L2 Egr1–/– and N3.L2 Egr1+/+ mice was significant (p < 0.05).

 
The peak of labeled cells in Fig. 3A is after the 9-day pulse period because cells labeled in the thymus continue to be exported to the periphery after labeling is stopped. We have found that thymectomized mice have very poor labeling of naive cells after a 9-day BrdU pulse (data not shown). Therefore, the reduced numbers of labeled 3.L2high cells in Egr1–/– mice during the first 20 days of the time course is either due to reduced thymic export or poor labeling in the thymus. We directly tested the ability of 3.L2high T cells to emigrate the thymus by performing intrathymic FITC injections on N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice. Both thymic lobes of age-matched N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were injected with FITC and 48 h later, peripheral lymphoid organs were surveyed for FITC+ RTE (26). Overall, N3.L2 Egr1–/– mice had 60% the number of 3.L2high RTE as littermate controls, whereas the number of 3.L2low RTE in N3.L2 Egr1–/– mice, and RTE in non-TCR transgenic Egr1–/– mice was comparable to their age-matched controls (Fig. 3B). Thus, it appears that Egr1 controls the number of RTE that accumulate in the periphery in a TCR-dependent manner.

N3.L2 Egr1-deficient mice have normal numbers of mature thymocytes

It is possible that Egr1 is regulating the ability of thymocytes to exit the thymus. However, the reduced numbers of RTE in N3.L2 Egr1–/– mice is apparent only on TCR transgenic backgrounds (Fig. 3), and there has been no requirement observed for TCR signaling in thymic egress. Furthermore, we have tested the ability of thymocytes from N.3L2 Egr1-deficient mice to migrate toward a sphingosine-1-phosphate (S1P) gradient. S1P has been shown to play a critical role in thymocyte egress from the thymus (27). Yet we found no difference in the efficiency of migration toward an S1P gradient in vitro between Egr1-deficient and wild-type cells (data not shown). Therefore, another explanation is needed for the TCR-dependent loss of RTE in N3.L2 Egr1–/– mice. As mentioned previously, Egr1-deficient mice have a defect in positive selection (5). It is possible that N3.L2 Egr1-deficient mice have fewer CD4SP cells available for export. This does not seem to be the case. Although impaired positive selection leads to a reduced percentage of 3.L2highCD4SP thymocytes in N3.L2 Egr1–/– mice, there are similar numbers of 3.L2highCD4SP thymocytes in N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice (Fig. 4A). This is because the loss of Egr1 leads to significantly increased thymic cellularity (5). The increased cellularity in Egr1-deficient thymuses is due to increased numbers of early double negative thymic precursors (F. J. Schnell and G. J. Kersh, manuscript in preparation).



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  		FIGURE 4. Normal numbers of mature CD4SP thymocytes in N3.L2 Egr1-deficient mice. A, Cell suspensions were prepared from thymuses of N3.L2 Egr1+/+ (n = 9) and N3.L2 Egr1–/– (n = 9) mice. Bar charts show total number of 3.L2highCD4SP thymocytes on either an Egr1+/+ (left) or Egr1–/– (right) background ± SEM. B, Single-cell suspensions of thymocytes were isolated from N3.L2 Egr1+/+ (left) and N3.L2 Egr1–/– (right) mice and stained with Abs against CD4, CD8, CD62L, and 3.L2 TCR. The contour plots display two representative stains for CD62L after gating on 3.L2highCD4+ thymocytes. Plots are representative of four independent experiments and seven to eight mice per group. C, The data show BrdU labeling of 3.L2high (left) and 3.L2low (right) CD4SP thymocytes from N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice given an i.p. injection of 1 mg of BrdU and sacrificed 5 h later. Data represent three to four mice per group ± SEM.

 
Once thymocytes make it through positive selection and up-regulate TCR expression and down-regulate either CD4 or CD8 to become SP cells, there are still multiple stages of development before thymocytes are able to exit the thymus. As SP thymocytes mature, they up-regulate CD62L before exiting the thymus (28). Therefore, the most mature thymocytes that are ready to emigrate the thymus are CD4SP TCRhighCD62Lhigh. We examined this subset and found that N3.L2 Egr1–/– mice have normal percentages of mature CD62Lhigh thymocytes (Fig. 4B). We have also used CD24 expression to assess the numbers of mature, premigrant thymocytes in N3.L2 Egr1–/– mice and again found no significant difference in the numbers of CD24lowCD4SP thymocytes between N3.L2 Egr1–/– and N3.L2 Egr1+/+ mice (data not shown). Therefore, even though positive selection is impaired in Egr1-deficient mice, the large size of the thymus in these mice produces a near normal number of mature thymocytes.

Another possibility that would explain the reduced numbers of RTE in 3.L2 transgenic Egr1–/– mice would be that the loss of Egr1 reduces the ability of premigrant thymocytes to proliferate. It has been shown by Campion et al. (29) that premigrant thymocytes proliferate in a TCR-dependent manner before leaving the thymus. The loss of Egr1 could lead to decreased numbers of thymocytes emigrating the thymus because of reduced proliferation before exiting the thymus. Therefore, we measured the in vivo proliferation of 3.L2highCD4SP thymocytes by doing a short-term BrdU pulse. N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were pulsed with BrdU and thymuses were harvested 5 h later to assess BrdU incorporation. As can be seen in Fig. 4C, 3.L2highCD4SP and 3.L2lowCD4SP Egr1–/– and Egr1+/+ thymocytes proliferated at comparable rates.

Impaired mature thymocyte survival in N3.L2 Egr1-deficient mice

Data from Fig. 4 show that N3.L2 Egr1–/– mice have normal numbers of functionally mature 3.L2highCD4SP thymocytes ready for export, yet N3.L2 Egr1–/– mice have reduced numbers of 3.L2high RTE. There are two explanations for this phenotype: the first being that 3.L2high Egr1-deficient thymocytes are not able to efficiently exit the thymus, or alternatively, 3.L2high Egr1-deficient RTE decay at a rapid rate once entering the periphery. To distinguish between these two possibilities, we directly assessed the ability of thymocytes to survive during the first days after introduction to the periphery. The experimental approach was to directly inject SP thymocytes i.v. into the bloodstream of immune-competent congenic mice and examine the accumulation of donor cells in peripheral lymphoid organs. By forcing thymocytes out of the thymus and into the periphery, we were able to directly determine whether the loss of RTE in N3.L2 Egr1–/– mice is related to a reduced ability to survive once in the periphery. Equal numbers of CD4SP thymocytes were injected into immune-competent congenic hosts and donor cell accumulation in host inguinal lymph nodes was assessed at days 1, 2, and 4. All previous experiments have shown that 3.L2low Egr1–/– cells behave in a similar manner to Egr1+/+ cells. Therefore, 3.L2low cells were used as an internal control in this experiment. As seen in Fig. 5A, 3.L2high Egr1–/– cells disappear more quickly when normalized to 3.L2low Egr1–/– cells. At day 1, 3.L2high and 3.L2low cells from N3.L2 Egr1–/– mice survived equally well (Fig. 5A). However, by day 2, 3.L2high Egr1–/– cells began to disappear at a higher rate than 3.L2low Egr1–/– cells, and this was even more pronounced at day 4 (Fig. 5A). We also examined donor cell accumulation in the spleen and found similar results (data not shown).



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  		FIGURE 5. Impaired survival of N3.L2 Egr1-deficient thymocytes. A, Thymocytes were isolated from age-matched N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice and i.v. injected into immune-competent congenic hosts. Inguinal lymph nodes were harvested 1, 2, and 4 days after injection to examine the accumulation of donor cells. Data represent the percentage survival of 3.L2high cells relative to 3.L2low cells at indicated time points. This number was determined by dividing the percentage of recovered CD4+ donor cells that were 3.L2high by the percentage of injected CD4SP thymocytes that were 3.L2high and then multiplying by 100. Results are presented as the average of three experiments ± SEM. B and C, Thymocytes from aged-matched N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were cultured in vitro for the indicated times. 3.L2high (B) and 3.L2low (C) CD4SP thymocyte viability was analyzed by staining for annexin V. The percentage of viability represents the percentage of annexin V-negative cells. Data represent the average of four to six mice per group analyzed over three independent experiments ± SEM. D, Thymocytes from aged-matched N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice were stained directly ex vivo for annexin V, CD4, CD62L, and 3.L2 TCR. Bar charts display the percentage of 3.L2highCD4+CD62high (left) and 3.L2highCD4+CD62low (right) cells that were positive for annexin V staining. Data represent five mice per group ± SEM. Significance was determined by an unpaired two-tailed Student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.0005).

 
These data indicate that the reduced numbers of 3.L2high RTE in N3.L2 Egr1-deficient mice is due to a problem with the short-term survival ability of 3.L2high thymocytes once shuttled into the periphery. Within 48 h of exiting the thymus, almost half of the 3.L2high RTE in N3.L2 Egr1–/– mice have disappeared (Figs. 3B and 5A). As mentioned previously, TCR signals are required for long-term naive T cell survival (9, 10, 11, 12, 13). Although naive T cells are able to survive for 1–2 wk after the loss of TCR signaling (9, 12), the survival of RTE could be regulated in a different manner. It is possible that RTE require a TCR-mediated signal that activates Egr1 within the first 48 h of entering the periphery to survive long-term. To address this issue, we removed both IL-7 and MHC signals and examined the ability of donor cells to survive in the periphery. IL-7R{alpha}-deficient CD4SP thymocytes directly injected into the bloodstream of class II MHC-deficient mice survived for 4 days as well as SP thymocytes that received either an IL-7 signal or class II MHC signal, or both signals (data not shown). RTE therefore do not seem to need either an IL-7 or MHC signal to survive over the first 4 days in the periphery, even though longer survival probably does require some combination of these signals (7, 8, 9, 10, 11, 12, 13).

The above data suggest that 3.L2highCD4SP Egr1–/– thymocytes are intrinsically less able to survive, either as they mature in the thymus, or once entering the periphery. To determine whether 3.L2high Egr1–/– thymocytes are inherently more prone to apoptosis, we cultured thymocytes from N3.L2 Egr1–/– and N3.L2 Egr1+/+ mice in vitro for 24 h and measured the percentage of viable cells remaining at various time points. 3.L2high Egr1–/– thymocytes displayed a faster rate of death compared with control thymocytes and compared with 3.L2low Egr1–/– thymocytes (Fig. 5, B and C). On average, only 41 ± 4% of the 3.L2high Egr1–/– thymocytes were viable at 24 h, yet 62 ± 3% of the 3.L2high Egr1+/+ thymocytes were viable at the same time point (Fig. 5B). The above results suggest that the N3.L2 Egr1–/– thymocytes that are able to make it through positive selection are not as fit as normal thymocytes and have a short-term survival defect.

We next sought to determine at what stage 3.L2high Egr1-deficient thymocytes were more prone to apoptosis. Gating on different subpopulations directly ex vivo, it appears that the CD62Lhigh population of thymocytes has a higher apoptosis rate in N3.L2 Egr1–/– mice (Fig. 5D). The CD62Llow subsets of 3.L2highCD4+ thymocytes in N3.L2 Egr1+/+ and N3.L2 Egr1–/– mice have similar rates of apoptosis (10.4 ± 2.1 vs 10.6 ± 2.3%), whereas the more mature CD62Lhigh population had very high rates of apoptosis in N3.L2 Egr1–/– mice (6.9 ± 2.0% for N3.L2 Egr1+/+ vs 20.0 ± 3.8% for N3.L2 Egr1–/–). It is somewhat surprising that 3.L2high Egr1–/– thymocytes do not show increased rates of apoptosis until the most mature stage of thymocyte development. The elevated numbers of DP cells in Egr1–/– mice can produce fairly normal numbers of SP cells even though the selection is inefficient, and the cells that do get positively selected behave normally during the initial stages postpositive selection (Fig. 4). However, when the 3.L2highCD4SP Egr1–/– thymocytes progress to the most mature stage of development, the CD62Lhigh stage, they begin to display a survival defect. Surprisingly, these alterations in survival that occur in the absence of Egr1 are TCR-dependent. It seems that non-TCR transgenic and 3.L2low Egr1–/– thymocytes are able to rearrange a TCR that is able to transmit a strong enough positive selection signal to bypass Egr1 and promote the survival of CD62Lhigh thymocytes and RTE. However, when the TCR is fixed by the presence of a TCR transgene, the required positively selecting signals that promote SP thymocyte and RTE survival are not fully transmitted because of the absence of Egr1. This result implies that positive selection signals are required not only to rescue cells from "death by neglect," but also for survival further down the thymocyte developmental pathway and even for the survival of cells after entering the periphery.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we have investigated the role of Egr1 in maintaining peripheral T cell homeostasis. By analyzing Egr1-deficient mice on a 3.L2 TCR transgenic background, we have found that Egr1 contributes to the maintenance of normal numbers of naive T cells circulating in the periphery. Examination of peripheral T cell numbers in N3.L2 Egr1–/– and OT-1 Egr1–/– mice revealed a significant decrease in the number of naive TCR transgenic CD4 and CD8 T cells, respectively. Further analysis of naive T cells in N3.L2 Egr1-deficient mice revealed no apparent defects in the proliferation or long-term survival of naive T cells. We did find, however, that N3.L2 Egr1-deficient mice have reduced numbers of RTE. Using BrdU labeling and intrathymic FITC injections, we were able to show that N3.L2 Egr1–/– mice have 40–50% fewer RTE accumulating in the periphery than age-matched controls. The reduced numbers of RTE in N3.L2 Egr1–/– mice can be traced back to increased apoptosis of thymocytes just before exiting the thymus and directly after entering the periphery.

As mentioned previously, Egr1 is required for efficient positive selection. Thus, the low numbers of RTE could be explained simply by a 3- to 4-fold reduction in the percentage of mature thymocytes in N3.L2 Egr1–/– mice. However, there are several pieces of information that argue against this. Firstly, although the percentage of mature thymocytes is low in Egr1–/– animals, the absolute numbers are only slightly reduced due to the abnormally large size of Egr1–/– thymuses (5) (Fig. 4A). Although the numbers of mature thymocytes produced in N3.L2 Egr1–/– mice is small compared with the large numbers of DP cells, the overall number of mature SP thymocytes is not dramatically reduced compared with a N3.L2 Egr1+/+ mouse. Furthermore, it has been demonstrated that it takes a very large difference in the number of mature thymocytes available for export to have a significant influence on the number of RTE. Almeida et al. (30) showed that mice that had a 90% reduction in the number of mature thymocytes only had a 3-fold decrease in the number of RTE. This finding suggests that the 40–50% reduction in RTE numbers that we observe in peripheral lymphoid organs of N3.L2 Egr1-deficient mice (Fig. 3B) would require a much more substantial decrease in absolute numbers of 3.L2highCD4SP thymocytes than we observe. We did detect a slight reduction in the numbers of 3.L2highCD4+ Egr1-deficient thymocytes at the CD62Lhigh stage, presumably from the increased rate of apoptosis we observed in this subset (Fig. 5D). However, the slight decrease in the CD62Lhigh subset was not statistically significant, and based on the Almeida et al. (30) study, the decrease we observed should not have a profound impact on the rate of thymic emigration.

Furthermore, our evidence suggests that near normal numbers of N3.L2 Egr1-deficient thymocytes are exported from the thymus. We have tested the ability of Egr1–/– thymocytes to migrate toward an S1P gradient, and found no differences in the efficiency of migration between N.3L2 Egr1+/+ and N.3L2 Egr1–/– CD4SP thymocytes (data not shown). Our findings in Fig. 5A similarly refute the idea that N3.L2 Egr1-deficient thymocytes have trouble exiting the thymus and homing to lymphoid organs. When we forced Egr1+/+ and Egr1–/– thymocytes into the periphery by i.v. injection, we eliminated differences in the ability to exit thymus. In this experiment, 3.L2high Egr1-deficient RTE migrated to peripheral lymphoid organs and survived nearly as well as control RTE over the first 24 h (Fig. 5A). However, by day 2, 3.L2high Egr1-deficient RTE began to decay at higher rates than control cells, and after 4 days of residing in the periphery, 3.L2high Egr1-deficient RTE numbers were reduced 40–50% compared with controls (Fig. 5A). The recovery of normal numbers of 3.L2high Egr1–/– cells from both the spleen and inguinal lymph nodes at day 1 suggests that the CD62Lhigh SP thymocytes in N3.L2 Egr1-deficient mice are exiting the thymus normally, and have no problem homing to peripheral lymphoid organs. The rapid decay of 3.L2high Egr1-deficient RTE over the next couple days implies that these cells have a survival defect.

Is the poor survival of Egr1-deficient RTE the result of an inability to transduce a TCR-dependent survival signal in the absence of Egr1? This seems unlikely. Previous studies have shown no need for RTE to compete for survival factors during the first week of entering the periphery. Berzins et al. (31) have shown that there is a 3-wk window in which RTE are exempt from having to compete for survival factors. It has also been shown that thymocytes injected into class II MHC-deficient or sufficient hosts are able to survive equally well regardless of the environment (32). Furthermore, we found that the removal of both survival factors IL-7 and class II MHC from CD4+ RTE does not affect their survival within the first 4 days of entering the periphery (data not shown). It seems that RTE are able to survive for at least 4 days while circulating in the periphery without much need for any of the known survival signals, certainly not TCR-dependent signals. And yet, almost half of 3.L2high Egr1-deficient CD4+ cells disappear after 2–4 days of circulating in the periphery, whereas cells that have rearranged endogenous TCR survive normally (Figs. 3B and 5A). Therefore, 3.L2high Egr1-deficient cells have decreased levels of survival at the most mature stage of development in the thymus and during the first days after entering the periphery, two stages of development that traditionally have been associated with very high rates of survival. That Egr1-deficient cells exhibit increased rates of apoptosis only when the TCR is fixed and only during stages that do not require much competition for survival factors leads us to conclude that cells undergoing positive selection via the 3.L2 or OT-1 TCR take on an Egr1-dependent program for survival that lasts until at least 4 days after thymic emigration.

How could the quality of the positive selection signal affect the survival of a cell that has matured and already migrated into the periphery? Presumably positive selection rescues DP cells from death and then also confers a type of grace period whereby selected cells are free from the rigors of competitive survival that take place among more mature peripheral T cells. It is estimated that it takes DP cells up to a week to complete positive selection and emigrate after first encountering a positive selection signal (33, 34). It follows then, that the grace period conferred by positive selection lasts at least 7–10 days, and could be longer, considering the evidence from Berzins et al. (31) that RTE are excluded from competition for survival factors for three weeks after exit from the thymus. This process ensures that there is a reasonable amount of turnover in the peripheral T cell pool, and that RTE are incorporated into the T cell repertoire throughout life. This Egr1-dependent grace period could be mediated by control of prosurvival gene expression. Indeed, we have found that DP thymocytes from Egr1-deficient mice have impaired Bcl-2 induction in response to TCR signals (5). However, the thymocytes that do make it to the status of mature SP in Egr1-deficient animals have the same levels of Bcl-2 as normal SP cells (data not shown). Thus, although there may be some defect in the initial up-regulation of Bcl-2 that makes positive selection inefficient, defective Bcl-2 expression does not seem to be the cause of poor RTE survival in Egr1–/– animals. It has also been suggested that positively selected thymocytes down-modulate certain genes that hinder their survival, such as Cerk and Tcl-30 (35). It is possible that Egr1 is required for the down-regulation of some proapoptotic genes. Further studies will need to be conducted to elucidate what survival and apoptotic genes are regulated by Egr1 in response to positive selection signals.

Collectively, these findings strongly suggest that the quality of signals produced during positive selection can impact the survival of mature thymocytes as they exit the thymus and attempt to integrate into the peripheral T cell pool. This model is much like the model proposed by Kieper et al. (36) in which the strength of the TCR signal received by naive T cells through contact with self-peptide-MHC complexes in the periphery determines the ability of T cells to survive. It is possible that thymocytes positively selected with high-affinity TCR interactions that are just below the negative selection threshold have a survival advantage at the mature SP stage and as RTE. The thymocytes that were selected with high affinity interactions would then be able to integrate into the peripheral T cell pool better because of an increased ability to survive during the first few days out of the thymus. In conclusion, we have found a novel role for positive selection and Egr1 in regulating the survival of mature thymocytes and recently emigrated thymocytes. Our results demonstrate that signals generated during positive selection are responsible not only for rescuing cells from death at the DP stage, but also in programming selected thymocytes for survival until they can become established in the periphery.


   Acknowledgments
 
We thank Jeffrey Milbrandt for the Egr1-deficient mice and Anna Bunin for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI-48784. Back

2 Address correspondence and reprint requests to Dr. Gilbert J. Kersh, Department of Pathology and Laboratory Medicine, Room 7311 Woodruff Memorial Building, Emory University, 101 Woodruff Circle, Atlanta, GA 30322. E-mail address: gkersh{at}emory.edu Back

3 Abbreviations used in this paper: Egr, early growth response gene; DP, double positive; SP, single positive; RTE, recent thymic emigrant; Hb, hemoglobin; S1P, sphingosine-1-phosphate. Back

Received for publication April 6, 2005. Accepted for publication June 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Milbrandt, J.. 1987. A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 238: 797-799. [Abstract/Free Full Text]
  2. Sukhatme, V. P., X. M. Cao, L. C. Chang, C. H. Tsai-Morris, D. Stamenkovich, P. C. Ferreira, D. R. Cohen, S. A. Edwards, T. B. Shows, T. Curran, et al 1988. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 53: 37-43. [Medline]
  3. Shao, H., D. H. Kono, L. Y. Chen, E. M. Rubin, J. Kaye. 1997. Induction of the early growth response (Egr) family of transcription factors during thymic selection. J. Exp. Med. 185: 731-744. [Abstract/Free Full Text]
  4. Bain, G., C. B. Cravatt, C. Loomans, J. Alberola-Ila, S. M. Hedrick, C. Murre. 2001. Regulation of the helix-loop-helix proteins, E2A and Id3, by the Ras-ERK MAPK cascade. Nat. Immunol. 2: 165-171. [Medline]
  5. Bettini, M., H. Xi, J. Milbrandt, G. J. Kersh. 2002. Thymocyte development in early growth response gene 1-deficient mice. J. Immunol. 169: 1713-1720. [Abstract/Free Full Text]
  6. Bettini, M., H. Xi, G. J. Kersh. 2003. T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms. Eur. J. Immunol. 33: 3109-3116. [Medline]
  7. Jameson, S. C.. 2002. Maintaining the norm: T-cell homeostasis. Nat. Rev. Immunol. 2: 547-556. [Medline]
  8. Sprent, J., C. D. Surh. 2003. Cytokines and T cell homeostasis. Immunol. Lett. 85: 145-149. [Medline]
  9. Seddon, B., R. Zamoyska. 2002. TCR signals mediated by Src family kinases are essential for the survival of naive T cells. J. Immunol. 169: 2997-3005. [Abstract/Free Full Text]
  10. Witherden, D., N. van Oers, C. Waltzinger, A. Weiss, C. Benoist, D. Mathis. 2000. Tetracycline-controllable selection of CD4+ T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules. J. Exp. Med. 191: 355-364. [Abstract/Free Full Text]
  11. Labrecque, N., L. S. Whitfield, R. Obst, C. Waltzinger, C. Benoist, D. Mathis. 2001. How much TCR does a T cell need?. Immunity 15: 71-82. [Medline]
  12. Polic, B., D. Kunkel, A. Scheffold, K. Rajewsky. 2001. How {alpha}{beta} T cells deal with induced TCR {alpha} ablation. Proc. Natl. Acad. Sci. USA 98: 8744-8749. [Abstract/Free Full Text]
  13. Priatel, J. J., S. J. Teh, N. A. Dower, J. C. Stone, H. S. Teh. 2002. RasGRP1 transduces low-grade TCR signals which are critical for T cell development, homeostasis, and differentiation. Immunity 17: 617-627. [Medline]
  14. Dorfman, J. R., I. Stefanová, K. Yasutomo, R. N. Germain. 2000. CD4+ T cell survival is not directly linked to self-MHC-induced TCR signaling. Nat. Immunol. 1: 329-335. [Medline]
  15. Ernst, B., D. S. Lee, J. M. Chang, J. Sprent, C. D. Surh. 1999. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 11: 173-181. [Medline]
  16. Goldrath, A. W., M. J. Bevan. 1999. Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts. Immunity 11: 183-190. [Medline]
  17. Kirberg, J., A. Berns, H. von Boehmer. 1997. Peripheral T cell survival requires continual ligation of the T cell receptor to major histocompatibility complex-encoded molecules. J. Exp. Med. 186: 1269-1275. [Abstract/Free Full Text]
  18. Viret, C., F. S. Wong, C. A. Janeway, Jr. 1999. Designing and maintaining the mature TCR repertoire: the continuum of self-peptide:self-MHC complex recognition. Immunity 10: 559-568. [Medline]
  19. Kieper, W. C., S. C. Jameson. 1999. Homeostatic expansion and phenotypic conversion of naive T cells in response to self peptide/MHC ligands. Proc. Natl. Acad. Sci. USA 96: 13306-13311. [Abstract/Free Full Text]
  20. Ebinu, J. O., D. A. Bottorff, E. Y. Chan, S. L. Stang, R. J. Dunn, J. C. Stone. 1998. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280: 1082-1086. [Abstract/Free Full Text]
  21. Lee, S. L., L. C. Tourtellotte, R. L. Wesselschmidt, J. Milbrandt. 1995. Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A. J. Biol. Chem. 270: 9971-9977. [Abstract/Free Full Text]
  22. Lee, S. L., Y. Sadovsky, A. H. Swirnoff, J. A. Polish, P. Goda, G. Gavrilina, J. Milbrandt. 1996. Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273: 1219-1221. [Abstract]
  23. Kao, H., P. M. Allen. 2005. An antagonist peptide mediates positive selection and CD4 lineage commitment of MHC class II-restricted T cells in the absence of CD4. J. Exp. Med. 201: 149-158. [Abstract/Free Full Text]
  24. Kersh, G. J., D. L. Donermeyer, K. E. Frederick, J. M. White, B. L. Hsu, P. M. Allen. 1998. TCR transgenic mice in which usage of transgenic {alpha}- and {beta}-chains is highly dependent on the level of selecting ligand. J. Immunol. 161: 585-593. [Abstract/Free Full Text]
  25. Curtsinger, J. M., D. C. Lins, M. F. Mescher. 1998. CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C–) to TCR/CD8 signaling in response to antigen. J. Immunol. 160: 3236-3243. [Abstract/Free Full Text]
  26. Scollay, R. G., E. C. Butcher, I. L. Weissman. 1980. Thymus cell migration. quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur. J. Immunol. 10: 210-218. [Medline]
  27. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427: 355-360. [Medline]
  28. Rosen, H., C. Alfonso, C. D. Surh, M. G. McHeyzer-Williams. 2003. Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist. Proc. Natl. Acad. Sci. USA 100: 10907-10912. [Abstract/Free Full Text]
  29. Le Campion, A., B. Lucas, N. Dautigny, S. Léaument, F. Vasseur, C. Pénit. 2002. Quantitative and qualitative adjustment of thymic T cell production by clonal expansion of premigrant thymocytes. J. Immunol. 168: 1664-1671. [Abstract/Free Full Text]
  30. Almeida, A. R., J. A. Borghans, A. A. Freitas. 2001. T cell homeostasis: thymus regeneration and peripheral T cell restoration in mice with a reduced fraction of competent precursors. J. Exp. Med. 194: 591-599. [Abstract/Free Full Text]
  31. Berzins, S. P., D. I. Godfrey, J. F. Miller, R. L. Boyd. 1999. A central role for thymic emigrants in peripheral T cell homeostasis. Proc. Natl. Acad. Sci. USA 96: 9787-9791. [Abstract/Free Full Text]
  32. Grandjean, I., L. Duban, E. A. Bonney, E. Corcuff, J. P. Di Santo, P. Matzinger, O. Lantz. 2003. Are major histocompatibility complex molecules involved in the survival of naive CD4+ T cells?. J. Exp. Med. 198: 1089-1102. [Abstract/Free Full Text]
  33. Huesmann, M., B. Scott, P. Kisielow, H. von Boehmer. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell 66: 533-540. [Medline]
  34. Lucas, B., F. Vasseur, C. Penit. 1994. Production, selection, and maturation of thymocytes with high surface density of TCR. J. Immunol. 153: 53-62. [Abstract]
  35. Mick, V. E., T. K. Starr, T. M. McCaughtry, L. K. McNeil, K. A. Hogquist. 2004. The regulated expression of a diverse set of genes during thymocyte positive selection in vivo. J. Immunol. 173: 5434-5444. [Abstract/Free Full Text]
  36. Kieper, W. C., J. T. Burghardt, C. D. Surh. 2004. A role for TCR affinity in regulating naive T cell homeostasis. J. Immunol. 172: 40-44. [Abstract/Free Full Text]



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