Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Aug 11.
Published in final edited form as: J Immunol. 2010 Dec 27;186(3):1343–1347. doi: 10.4049/jimmunol.1002238

Post-selection thymocyte maturation and emigration are independent of IL-7 and ERK5

Michael A Weinreich 1, Stephen C Jameson 1, Kristin A Hogquist 1
PMCID: PMC4980999  NIHMSID: NIHMS753477  PMID: 21187442

Abstract

The transcription factor Kruppel-Like Factor 2 (KLF2) controls the emigration of conventional T cells from the thymus through its regulation of the cell surface receptor, S1P1. Prior to KLF2 expression, developing T cells require a positive selection signal through the T cell receptor. However, following positive selection there are time, spatial and maturational events that occur before KLF2 is finally upregulated and emigration occurs. We are interested in determining the signals that upregulate KLF2 and allow thymocytes to emigrate into circulation, and if they are linked to functional maturation. In endothelial cells KLF2 expression has been shown to be dependent on the MAP kinase ERK5. Furthermore, it has been reported that IL-7 signaling leads to the phosphorylation of ERK5. Thus it was hypothesized that IL-7 receptor signaling through ERK5 could drive the expression of KLF2. In this study we provide evidence that this hypothesis is incorrect. We also found that CD8 lineage specification occurred normally in the absence of IL-7R signaling, in contrast to a recently proposed model. We showed that both CD4 and CD8 T cells complete maturation and express KLF2 independently of ERK5 and IL-7.

Introduction

T cells develop in ordered differentiation stages within the thymus. These stages can be differentiated by expression of T cell receptor (TCR) coreceptors, CD4 and CD8. The most immature progenitors, the double negative (DN) thymocytes, express neither CD4 nor CD8. During the DN stage of selection, expression of the β chain of the TCR occurs. At this stage the thymocytes proliferate and their survival is dependent on the cytokine IL-7. Thymocytes then express both CD4 and CD8, signifying the double positive (DP) stage.

Positive selection of DP thymocytes is marked by high levels of the TCR and upregulation of CD69 and CCR7 on the cell surface. Following positive selection, the DP thymocytes down-regulate one of their coreceptors in a manner that is dependent on the class of the selective MHC. Those selected on Class II MHC become CD4 single positive (SP) thymocytes and those selected on Class I become CD8 SPs. Thymocytes transitioning from the DP to SP stage migrate to the medulla, dependent on CCR7-mediated chemotaxis.

The maturation state of the SP population can be further differentiated using additional cell surface markers. Heat stable antigen (HSA) and CD69 are highly expressed post-selection and on semi-mature SPs. HSA and CD69 are down regulated with maturation. The opposite pattern is observed with Qa-2 and CD62L, as expression increases with maturation(1). The maturation is not only a superficial change in surface receptors but also functional. Kishimoto and Sprent demonstrated that TCR stimulation of semi-mature (HSA high) SPs induces death while mature thymocytes respond by proliferating(2). In other words, semi-mature SPs remain susceptible to negative selection.

Time spent in the medulla is important to allow interactions between semi-mature thymocytes and the unique medullary stroma. Some tissue specific antigens are expressed only by medullary thymic epithelial cells, and depend on the transcription factor Aire (autoimmune regulator) (3). The Aire gene was originally discovered as mutated in human patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (4). Additionally, Takahama and colleagues have demonstrated that thymocytes from mice deficient in the chemokine receptor CCR7 do not travel to the thymic medulla and these thymocytes are reactive to self-antigens (5, 6). More recently the Cyster group found that forcing thymocytes to emigrate from the thymus early, with transgenic expression of S1P1, led to an increase in lymphoid infiltrates in tissues(7). All of these findings support an important role for allowing negative-selection susceptible thymocytes to survey the thymic medulla.

The transcription factor Kruppel-like factor 2 (KLF2) is required for T cells to emigrate from the thymus via its role in regulating the receptor S1P1(8). To better understand the mechanisms that control medullary residency of thymocytes we investigated the regulation of KLF2. Positive selection is an important checkpoint for thymocyte development prior to KLF2 expression. Since KLF2 is not expressed until a minimum of two days after positive selection and after migration from the thymic cortex to the medulla occurs(9), we felt that it was unlikely that positive selection directly induces KLF2 expression.

The cytokine IL-7 is necessary for thymocyte and T cell survival(10). Signaling through the IL-7 receptor is necessary for the survival of DNs(11). However at the DP stage the IL-7R is not expressed and DPs are refractory to cytokine signaling because of expression of the signaling suppressor SOCS1 (12). Furthermore, the major source of IL-7 in the thymus is the cortical-medullary junction and inside the medulla(13). In addition, IL-7 can induce the expression of KLF2 following TCR stimulation induced down-regulation of KLF2(14). We speculated that if KLF2 is dependent on IL-7 signaling this would explain the delayed expression following positive selection, and would ensure that SP thymocytes did not emigrate from the organ until they spent at least some time surveying the medulla.

If IL-7 was implicated in the regulation of KLF2 then what signaling pathway would this work through? Erk5 is an important regulator of KLF2 in other cell types (15, 16). Erk5 is a member of the mitogen-activated protein kinase (MAPK) family. Erk5 activates the transcription factor myocyte enhancer factor 2 (MEF2)(15). Mice deficient in either Erk5 or KLF2 die embryologically of angiogenic defects(17, 18). Winoto and colleagues demonstrated MEF2 can bind the KLF2 promoter and that knockdown of Erk5 reduces the expression of KLF2 in T cells(15). Importantly, this group reported that T cell stimulation with IL-7 led to the phosphorylation of ERK5, leading to the hypothesis that IL-7 signals the upregulation of KLF2 in T cells(15).

In this study we sought to test the hypothesis that post-positive selection signaling by IL-7 through ERK5 leads to the expression of KLF2 and resulting emigration of thymocytes. We found no evidence for IL-7 regulation of KLF2 using genetically deficient cells or in vivo IL-7R blockade. We also found that genetically ERK5 deficient T cells underwent normal development and showed no deficiency in KLF2. Our findings lead us to conclude that IL-7 and ERK5 do not control KLF2 expression or the semi-mature to mature SP thymocyte transition.

Materials and Methods

Mice

IL-7 receptor KO mice have been described (23) and were provided by M. Farrar (University of Minnesota, Minneapolis, MN). KLF2-GFP reporter mouse was generated by our laboratory and described previously (21). Erk5fl/fl mice were provided by Cathy Tournier (University of Manchester, Manchester, UK) (26). Erk5fl/fl mice were bred to CD4-cre mice obtained from Taconic farms at our facility. All mice were treated in accordance with federal guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee.

In vivo IL-7R and S1P1 blockade

Anti-IL-7R (A7R34) was purified in our laboratory from the hybridoma. The antibody was injected at 1 mg/mouse intraperitoneally (i.p.) every two days. S1P receptor agonist (Merck, Rahway, NJ) was provided to us by Marc Jenkins, University of Minnesota. This was injected 1 mg/kg daily i.p.

Flow cytometry

All directly conjugated antibodies were purchased from BD Bioscience, eBioscience, Invitrogen, or BioLegend. For surface staining, cells were incubated with antibodies in PBS, with 1% FCS, and 0.02% azide for 30 minutes on ice. For intracellular staining for phospho-STAT5, cells were fixed and permeabilized in methanol according to (29). An APC conjugated Goat anti-RatIgG secondary antibody was used to detect A7R34 (rat IgG2a) binding to thymocytes in vivo. In this experiment, SP thymocytes were identified by their higher MHC Class I expression using an antibody to Kb (Y3, mouse IgG2b) to avoid cross-reactivity on rat antibodies used to detect CD4 and CD8. Data was collected on a LSR-II cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.).

BrdU labeling

BrdU labeling and staining was performed as previously described (9). 100 µL of 10 mg/mL BrdU (Sigma-Aldrich; B5002) was injected i.p. twice at a 4-hour interval. Mice were harvested and analyzed 8 days later. Thymus and lymph node cells were cell surface stained as described. Then intracellular staining was done using the APC BrdU flow kit (BD Biosciences), substituting the anti-BrdU antibody (PRB-1) from Phoenix Flow Systems.

Cell Sorting

Conventional SP thymocytes were purified by on a FACSVantage (Becton Dickinson) by gating on CD25, γδ TCR, NK1.1 negative cells then selecting the CD4 and CD8 SP populations. 15 IL-7R KO thymi were pooled for this experiment. For mature CD4 SP thymocytes, Qa2 positive cells were sorted.

RNA purification and analysis

RNA was purified using the RNeasy kit (Qiagen). cDNA was generated using SuperScriptII Platinum Two-Step qRT-PCR kit (Invitrogen). qPCR was performed on a SmartCycler real-time PCR machine (Cepheid) using FastStart SYBR Green Master Mix kit (Roche).

Results

KLF2 is expressed in IL-7 receptor deficient T cells

We set out to test the hypothesis that KLF2 expression in T cells was dependent on post-positive selection IL-7 signals. Consistent with published reports(9, 19) we show that neither IL-7 receptor nor KLF2 are expressed in DP thymocytes. IL-7R is expressed directly after positive selection in the “youngest” SP thymocytes (as determined by HSA and CD69) and continues to be expressed in all subsequent thymocyte subsets. Ex vivo analysis of STAT5 phosphorylation supports this pattern of IL-7 responsiveness(20). In contrast KLF2 is only expressed in more mature SP subsets(9). We find that IL-7R expression precedes KLF2 in SP thymocytes using a KLF2 reporter mouse that expresses a KLF2-GFP fusion protein(21)(Fig. 1). Since post-selection thymocytes migrate to the cytokine rich medulla, dependent on CCR7(5, 22), we conclude that developing thymocytes are likely to be receiving signals through the IL-7R prior to expression of KLF2.

Figure 1. IL-7 receptor precedes KLF2 expression after positive selection.

Figure 1

Open in a new tab

IL-7 receptor and GFP (KLF2) expression in thymocytes from an unmanipulated KLF2-GFP reporter mouse, after gating on DP, or “young”, “middle-aged”, and “old” CD4 SP as determined by HSA and CD69 staining. n =3

Next, to test the dependence on IL-7 for KLF2 expression we analyzed Il7ra−/− mice. Il7ra−/− mice have greatly reduced thymocyte cell number due the role of IL-7 in survival of thymocytes at the DN stage(11, 23). However, as previously reported, we find a population of CD4 and CD8 SP thymocytes that develop in these mice (Fig. 2A). Because of the low cell numbers we focused our analysis on the more prevalent CD4 SP population but we observed similar trends in CD8 SP (data not shown). To attempt to eliminate error based on comparing altered subsets within the SP thymocyte population, we limited our analysis to conventional αβ T cells by excluding regulatory, γδ, and NK T cells by gating out CD25, γδ TCR, and NK1.1+ cells. Since KLF2 is necessary for emigration from the thymus, if IL-7R deficient SPs were deficient in KLF2 expression we would expect thymocyte retention of mature SP. Indeed, the CD4 SPs from the Il7ra−/− thymus appeared more mature (HSA low, Qa2 high) consistent with retention, though CD69 and CD62L, which also change with SP maturation, were not as severely affected (Fig. 2B).

Figure 2. KLF2 is expressed in IL-7R KO thymocytes.

Figure 2

Open in a new tab

A, CD4 by CD8 profile of control and IL-7R KO thymi. B, CD69, CD62L, HSA, and Qa2 expression on Dump (CD25, γδ TCR, NK1.1) negative, CD4 SP thymocytes. n > 5 C, KLF2 mRNA expression from indicated cell sorted thymus subsets. Fold change was standardized to β catenin and graphed relative to expression in WT DP thymocytes, which was set to 1. Cells were sorted from 1 WT B6 and 15 pooled Il7ra−/− mice.

To directly measure KLF2 expression in IL-7R deficient mice, we pooled thymocytes from 15 Il7ra−/− mice and sorted DP, CD4 SP and CD8 SP cells. In contrast to the mature cell surface phenotype, which is consistent with decreased KLF2 expression, quantitative analysis of KLF2 mRNA in these subsets compared to WT thymocytes showed similar levels of expression (Fig. 2C). These findings indicate that the few mature thymocytes that develop in IL-7R deficient mice are able express KLF2, arguing against an absolute requirement for IL-7R signals.

Thymocyte emigration does not depend on IL-7 receptor signals

Since development for the vast majority of thymocytes is dependent on IL-7, we considered the possibility that our findings with IL-7R deficient thymocytes were not representative of what occurs when thymocytes develop normally with IL-7 receptor signaling. To allow thymocytes to develop normally through the DP stage then block the IL-7R, we devised a short-term antibody blockade strategy (Fig. 3A). We limited the blockade to 6 days to minimize the complications from effects of the blockade on the DN population subsequently entering the SP populations. As an emigration blockade positive control, we treated mice with an S1P1 agonist(24).

Figure 3. IL-7R blockade does not prevent thymocyte emigration.

Figure 3

Open in a new tab

A, Experimental design and dosing schedule for IL-7R receptor and S1P1 blockade. To test the efficacy of IL-7R blockade in vivo, C57BL/6 mice were injected i.p. with 1 mg anti-IL-7R (A7R34) (solid line) or isotype control (Rat IgG2a) (shaded histogram) and evaluated 48 hours later. B, Analysis of Kb+ (CD4 and CD8 SP) thymocytes with a secondary antibody (APC Goat anti-RatIgG). C, Thymocytes from anti-IL-7R treated mice (bottom histogram) or isotype control treated mice (top histogram) were stimulated with 25ng/ml IL-7 in vitro. After 20 minutes, intracellular staining was performed with an antibody to phospho-STAT5. Histograms show CD4SP thymocytes. n= 4 animals per group. D, Total thymus cell number in treated and control mice. E, CD4 by CD8 thymus profile of representative treated and control mice. F, CD4 SP number in treated and control mice. G, Enumeration of BrdU-labeled cells in the dump-, CD4 SP gate. H, Ratio of CD4 to CD8 SP in the treated and control mice. Immature CD8SP were excluded from the analysis. Control and IL-7R blockade: n=3.

First, we evaluated if the in vivo blockade of IL-7R was effective. Using a goat anti-rat secondary antibody, we observed staining of IL-7R+ (SP) thymocytes in mice treated with ant-IL-7R, but not isotype control (Fig. 3B). Anti-IL-7R treated mice also showed no detectable IL-7 dependent phosphorylation of STAT5 in vitro (Fig. 3C). In addition, the total thymocyte number decreased approximately 50 percent with this short-term IL-7R antibody treatment (Fig. 3D). Together these data indicate that the antibody effectively reached the thymus and blocked IL-7R signaling.

We found that IL-7R blockade led to only a modest increase in the percentage (Fig. 3E) and no increase in the total number (Fig. 3F) of SP thymocytes. An increase in SP thymocyte numbers would be expected with a thymocyte emigration defect, illustrated by the effect of an S1P1 inhibitor (Fig. 3E,F). Nor did acute IL-7R blockade alter the maturation phenotype (HSA, Qa2) of SP thymocytes (data not shown), suggesting that the changed phenotype in IL-7R deficient mice could have been due to enhanced thymic recirculation of peripheral T cells, possibly secondary to lymphopenia in those animals. To avoid the confounding effect of anti-IL-7R at the DN stage that resulted in reduced cellularity, we incorporated a BrdU pulse-labeling step (Fig. 3A). This allowed us to label a cohort of proliferating DP thymocytes, allowing them to develop with normal IL-7R signaling and then administered the IL-7R or S1P1 blockade for 6 days. Again the S1P1 inhibitor led to an accumulation of labeled cells while IL-7R blockade did not (Fig. 3G). Analysis of IL-7R genetically deficient thymocytes and in vivo blockade of IL-7R did not support a role for IL-7R in KLF2 expression and thymic emigration. Importantly, we also did not observe an alteration in the CD4 to CD8 SP ratio (Fig. 3H), suggesting that IL-7 does not play an obligatory role in the lineage commitment or survival of CD8 T cells.

Redundant function of ERK5 in T cell development

The ERK5 knockout mouse is embryonically lethal from similar endothelial defects as the KLF2 knockout mouse (18). In addition it has been reported that ERK5 is required for KLF2 expression in mouse embryonic fibroblast cells and ERK5 knockdown in mature T cells decreased KLF2 expression (15). IL-7R signaling was shown to result in ERK5 phosphorylation in T cells (15). Although we did not observe an obligatory role for IL-7 in KLF2 expression in thymocytes, it is possible that redundant cytokines or other signals would activate ERK5 in thymocytes to induce KLF2 expression. To test the role of ERK5 in T cell development and expression of KLF2 in T cells, we obtained ERK5 floxed mice and bred them to CD4-cre mice to generate T cell specific ERK5 deficiency (25).

We found no significant change in CD4 or CD8 T cell number in the thymus, spleen, or lymph node (Fig. 4A and data not shown). Also we did not observe a difference in cell surface phenotype, including activation and migratory receptors, in the ERK5 deficient T cells (Fig. 4B). In intact animals or competitive mixed bone marrow chimeras, ERK5 deficient T cells did not differ in proliferation or memory cell surface phenotype, (data not shown). To investigate the relationship between ERK5 and KLF2, we sorted mature, CD4 SP thymocytes from WT and ERK5 deficient mice. While ERK5 was undetectable, KLF2 was expressed to similar levels in WT and ERK5 deficient T cells (Fig. 4). These results indicate that ERK5 is not necessary for T cell development and function.

Figure 4. ERK5 deficient T cells develop and express KLF2 normally.

Figure 4

Open in a new tab

A, Representative CD4 by CD8 thymocyte profiles, left two panels, and spleen, right, from CD4cre/Erk5fl/fl and littermate control mice. B, Cell surface phenotype of CD4 SP thymocytes, gray solid=littermate control and black line=CD4cre/Erk5fl/fl. C, Analysis of ERK5 and KLF2 mRNA from sorted Qa2-expressing, mature CD4 SP thymocytes. Fold change was standardized to HPRT and graphed relative to expression in WT mice. Error bar indicates standard deviation. nd=not detected

Discussion

After we generated this data, Arthur’s group published similar findings with ERK5 deficient T cells from conditionally deficient mice (26). They reported no defect in development for ERK5 deficient T cells and normal numbers of T cells in the spleen and lymph node. While no change was found in basal KLF2 levels in the periphery, the group did report lower KLF2 expression after TCR stimulation of ERK5 deficient T cells but this did not have functional consequence as no difference in KLF2 target genes, such as S1P1 and CD62L were observed. Taken together, ERK5 does not appear to play a critical, non-redundant role in T cell development.

Signaling through the IL-7 receptor on the other hand has been suggested to play a major role in the survival and function of T cells at multiple steps during development (10, 27). However, using two independent loss of function approaches we did not find an obligatory role for IL-7R in regulation of KLF2 expression. IL-7 has been shown to induce to the re-expression of KLF2 following activation(14). However, the maintenance of KLF2 in naïve T cells is IL-7 independent(28). Together this is consistent with IL-7 independent regulation of KLF2 during at least some stages of T cell differentiation.

We also did not observe a reduction in the proportion or number of CD8 SP thymocytes, as predicted by recent work suggesting that IL-7 signals carry out positive selection of CD8 T cells(27). However, that study employed a gain of function approach to suggest that IL-7 signals can drive CD8 T cell development, and did not test the hypothesis using loss of function approaches, such as we used here. Interestingly, CD8 T cell development did not occur in the absence of all γc cytokine signaling, which includes IL-7 (27). Thus it remains possible that IL-7 can contribute to CD8 survival and lineage commitment, but in its absence, other γc cytokines such as IL-4 can substitute.

It is worth noting here that the termination of RAG, and induction of migration to the medulla, which are key initial steps in positive selection initiated by the TCR signal, are not replaceable by either IL-7 or other γc cytokine signals, in contrast to the sentiment expressed in (27). Thus we would argue that positive selection is best understood as a multistep process consisting of: 1) initial survival, receptor specificity fixation, and migration activities in DP cells, followed immediately or concurrently by 2) signals (through cytokines in the case of CD8 T cells) that fix cytotoxic/helper lineage in emerging CD4/8 SP cells, and finalized by 3) a biochemical realignment of the TCR signaling apparatus to achieve proliferation competence, that happens as cells transition from semi-mature SP to mature SP. It is this final stage where KLF2 is upregulated, and which is still largely not understood in genetic/biochemical terms. Since large changes are occurring at this stage both in the functional response to TCR stimulation and migration pattern of these T cells, it is interesting to speculate that genome wide changes in chromatin structure underlie the biological effects. Another possibility would be altered microRNA expression, which can alter the expression of many genes simultaneously. The transition from semimature to functionally competent T cells is an important developmental step that is regulated by a yet undefined mechanism.

REFERENCES

  • 1.Weinreich MA, Hogquist KA. Thymic emigration: when and how T cells leave home. J Immunol. 2008;181:2265–2270. doi: 10.4049/jimmunol.181.4.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kishimoto H, Sprent J. Negative selection in the thymus includes semimature T cells. J Exp Med. 1997;185:263–271. doi: 10.1084/jem.185.2.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, von Boehmer H, Bronson R, Dierich A, Benoist C, Mathis D. Projection of an immunological self shadow within the thymus by the aire protein. Science. 2002;298:1395–1401. doi: 10.1126/science.1075958. [DOI] [PubMed] [Google Scholar]
  • 4.Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, Krohn KJ, Lalioti MD, Mullis PE, Antonarakis SE, Kawasaki K, Asakawa S, Ito F, Shimizu N. Positional cloning of the APECED gene. Nat Genet. 1997;17:393–398. doi: 10.1038/ng1297-393. [DOI] [PubMed] [Google Scholar]
  • 5.Ueno T, Saito F, Gray DH, Kuse S, Hieshima K, Nakano H, Kakiuchi T, Lipp M, Boyd RL, Takahama Y. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J Exp Med. 2004;200:493–505. doi: 10.1084/jem.20040643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kurobe H, Liu C, Ueno T, Saito F, Ohigashi I, Seach N, Arakaki R, Hayashi Y, Kitagawa T, Lipp M, Boyd RL, Takahama Y. CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity. 2006;24:165–177. doi: 10.1016/j.immuni.2005.12.011. [DOI] [PubMed] [Google Scholar]
  • 7.Zachariah MA, Cyster JG. Neural Crest-Derived Pericytes Promote Egress of Mature Thymocytes at the Corticomedullary Junction. Science. 2010 doi: 10.1126/science.1188222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Carlson CM, Endrizzi BT, Wu J, Ding X, Weinreich MA, Walsh ER, Wani MA, Lingrel JB, Hogquist KA, Jameson SC. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature. 2006;442:299–302. doi: 10.1038/nature04882. [DOI] [PubMed] [Google Scholar]
  • 9.McCaughtry TM, Wilken MS, Hogquist KA. Thymic emigration revisited. J Exp Med. 2007;204:2513–2520. doi: 10.1084/jem.20070601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Mazzucchelli R, Durum SK. Interleukin-7 receptor expression: intelligent design. Nat Rev Immunol. 2007;7:144–154. doi: 10.1038/nri2023. [DOI] [PubMed] [Google Scholar]
  • 11.Maraskovsky E, O'Reilly LA, Teepe M, Corcoran LM, Peschon JJ, Strasser A. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1−/− mice. Cell. 1997;89:1011–1019. doi: 10.1016/s0092-8674(00)80289-5. [DOI] [PubMed] [Google Scholar]
  • 12.Yu Q, Park JH, Doan LL, Erman B, Feigenbaum L, Singer A. Cytokine signal transduction is suppressed in preselection double-positive thymocytes and restored by positive selection. J Exp Med. 2006;203:165–175. doi: 10.1084/jem.20051836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Alves NL, Richard-Le Goff O, Huntington ND, Sousa AP, Ribeiro VS, Bordack A, Vives FL, Peduto L, Chidgey A, Cumano A, Boyd R, Eberl G, Di Santo JP. Characterization of the thymic IL-7 niche in vivo. Proc Natl Acad Sci U S A. 2009;106:1512–1517. doi: 10.1073/pnas.0809559106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Schober SL, Kuo CT, Schluns KS, Lefrancois L, Leiden JM, Jameson SC. Expression of the transcription factor lung Kruppel-like factor is regulated by cytokines and correlates with survival of memory T cells in vitro and in vivo. J Immunol. 1999;163:3662–3667. [PubMed] [Google Scholar]
  • 15.Sohn SJ, Li D, Lee LK, Winoto A. Transcriptional regulation of tissue-specific genes by the ERK5 mitogen-activated protein kinase. Mol Cell Biol. 2005;25:8553–8566. doi: 10.1128/MCB.25.19.8553-8566.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Young A, Wu W, Sun W, Larman HB, Wang N, Li YS, Shyy JY, Chien S, Garcia-Cardena G. Flow activation of AMP-activated protein kinase in vascular endothelium leads to Kruppel-like factor 2 expression. Arterioscler Thromb Vasc Biol. 2009;29:1902–1908. doi: 10.1161/ATVBAHA.109.193540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wani MA, Means RT, Jr, Lingrel JB. Loss of LKLF function results in embryonic lethality in mice. Transgenic Res. 1998;7:229–238. doi: 10.1023/a:1008809809843. [DOI] [PubMed] [Google Scholar]
  • 18.Regan CP, Li W, Boucher DM, Spatz S, Su MS, Kuida K. Erk5 null mice display multiple extraembryonic vascular and embryonic cardiovascular defects. Proc Natl Acad Sci U S A. 2002;99:9248–9253. doi: 10.1073/pnas.142293999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Jin R, Zhang J, Chen W. Thymic output: influence factors and molecular mechanism. Cell Mol Immunol. 2006;3:341–350. [PubMed] [Google Scholar]
  • 20.Van De Wiele CJ, Marino JH, Murray BW, Vo SS, Whetsell ME, Teague TK. Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation. J Immunol. 2004;172:4235–4244. doi: 10.4049/jimmunol.172.7.4235. [DOI] [PubMed] [Google Scholar]
  • 21.Weinreich MA, Takada K, Skon C, Reiner SL, Jameson SC, Hogquist KA. KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors. Immunity. 2009;31:122–130. doi: 10.1016/j.immuni.2009.05.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kwan J, Killeen N. CCR7 directs the migration of thymocytes into the thymic medulla. J Immunol. 2004;172:3999–4007. doi: 10.4049/jimmunol.172.7.3999. [DOI] [PubMed] [Google Scholar]
  • 23.Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB, Meyer JD, Davison BL. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med. 1994;180:1955–1960. doi: 10.1084/jem.180.5.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature. 2004;427:355–360. doi: 10.1038/nature02284. [DOI] [PubMed] [Google Scholar]
  • 25.Hayashi M, Kim SW, Imanaka-Yoshida K, Yoshida T, Abel ED, Eliceiri B, Yang Y, Ulevitch RJ, Lee JD. Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J Clin Invest. 2004;113:1138–1148. doi: 10.1172/JCI19890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ananieva O, Macdonald A, Wang X, McCoy CE, McIlrath J, Tournier C, Arthur JS. ERK5 regulation in naive T-cell activation and survival. Eur J Immunol. 2008;38:2534–2547. doi: 10.1002/eji.200737867. [DOI] [PubMed] [Google Scholar]
  • 27.Park JH, Adoro S, Guinter T, Erman B, Alag AS, Catalfamo M, Kimura MY, Cui Y, Lucas PJ, Gress RE, Kubo M, Hennighausen L, Feigenbaum L, Singer A. Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells. Nat Immunol. 2010;11:257–264. doi: 10.1038/ni.1840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Endrizzi BT, Jameson SC. Differential role for IL-7 in inducing lung Kruppel-like factor (Kruppel-like factor 2) expression by naive versus activated T cells. Int Immunol. 2003;15:1341–1348. doi: 10.1093/intimm/dxg133. [DOI] [PubMed] [Google Scholar]
  • 29.Vang KB, Yang J, Mahmud SA, Burchill MA, Vegoe AL, Farrar MA. IL-2,-7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development. J Immunol. 2008;181:3285–3290. doi: 10.4049/jimmunol.181.5.3285. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES