Postselection Thymocyte Maturation and Emigration Are Independent of IL-7 and ERK5

Michael A. Weinreich, Stephen C. Jameson and Kristin A. Hogquist
J Immunol February 1, 2011, 186 (3) 1343-1347; DOI: https://doi.org/10.4049/jimmunol.1002238

Abstract

The transcription factor Krüppel-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 TCR. 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 whether they are linked to functional maturation. In endothelial cells KLF2 expression has been shown to be dependent on the mitogen-activated protein kinase ERK5. Furthermore, it has been reported that IL-7 signaling leads to the phosphorylation of ERK5. Thus, we hypothesized that IL-7R 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.

T cells develop in ordered differentiation stages within the thymus. These stages can be differentiated by expression of 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 downregulate 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 Ag (HSA) and CD69 are highly expressed postselection and on semimature SPs. HSA and CD69 are downregulated 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 (2) demonstrated that TCR stimulation of semimature (HSAhigh) SPs induces death, whereas mature thymocytes respond by proliferating. In other words, semimature SPs remain susceptible to negative selection.

Time spent in the medulla is important to allow interactions between semimature thymocytes and the unique medullary stroma. Some tissue-specific Ags 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 (4). Additionally, Takahama and colleagues (5, 6) 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-Ags. More recently, Zachariah and Cyster (7) found that forcing thymocytes to emigrate from the thymus early, with transgenic expression of S1P1, led to an increase in lymphoid infiltrates in tissues. All of these findings support an important role for allowing negative selection-susceptible thymocytes to survey the thymic medulla.

The transcription factor Krüppel-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. Because KLF2 is not expressed until a minimum of 2 d after positive selection and after migration from the thymic cortex to the medulla occurs (9), we thought 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-7R 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). Additionally, IL-7 can induce the expression of KLF2 following TCR stimulation-induced downregulation of KLF2 (14). We speculated that if KLF2 is dependent on IL-7 signaling, this would explain the delayed expression following positive selection, and it would ensure that SP thymocytes did not emigrate from the organ until they spent at least some time surveying the medulla.

If IL-7 were 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 MAPK family. Erk5 activates the transcription factor myocyte enhancer factor 2 (15). Mice deficient in either Erk5 or KLF2 die embryologically of angiogenic defects (17, 18). Winoto and colleagues (15) demonstrated that myocyte enhancer factor 2 can bind the KLF2 promoter and that knockdown of Erk5 reduces the expression of KLF2 in T cells. 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 semimature to mature SP thymocyte transition.

Materials and Methods

Mice

IL-7R knockout (KO) mice have been described (19) and were provided by M. Farrar (University of Minnesota, Minneapolis, MN). KLF2-GFP reporter mouse was generated by our laboratory and has been described previously (20). Erk5fl/fl mice were provided by Cathy Tournier (University of Manchester, Manchester, U.K.) (21). Erk5fl/fl mice were bred to CD4-cre mice obtained from Taconic. 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 Ab was injected at 1 mg/mouse i.p. every 2 d. S1P1 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 Abs were purchased from BD Biosciences, eBioscience, Invitrogen, or BioLegend. For surface staining, cells were incubated with Abs in PBS, with 1% FCS, and 0.02% azide for 30 min on ice. For intracellular staining for phospho-STAT5, cells were fixed and permeabilized in methanol according to Vang et al. (22). An allophycocyanin-conjugated goat anti-rat IgG secondary Ab 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 Ab to Kb (Y3, mouse IgG2b) to avoid cross-reactivity on rat Abs used to detect CD4 and CD8. Data were collected on an LSR II cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

BrdU labeling

BrdU labeling and staining were performed as previously described (9). One hundred microliters 10 mg/ml BrdU (Sigma-Aldrich; B5002) was injected i.p. twice at a 4-h interval. Mice were harvested and analyzed 8 d later. Thymus and lymph node cells were cell surface stained as described. Then, intracellular staining was done using an allophycocyanin BrdU flow kit (BD Biosciences), substituting the anti-BrdU Ab (PRB-1) from Phoenix Flow Systems.

Cell sorting

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

RNA purification and analysis

RNA was purified using the RNeasy kit (Qiagen). cDNA was generated using a SuperScript II Platinum two-step quantitative RT-PCR kit (Invitrogen). Quantitative PCR was performed on a SmartCycler real-time PCR machine (Cepheid) using a FastStart SYBR Green Master Mix kit (Roche).

Results

KLF2 is expressed in IL-7R-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, 23) we show that neither the IL-7R nor KLF2 is 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 (24). 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 (20) (Fig. 1). Because postselection thymocytes migrate to the cytokine-rich medulla, dependent on CCR7 (5, 25), we conclude that developing thymocytes are likely to be receiving signals through the IL-7R prior to expression of KLF2.

FIGURE 1.
FIGURE 1.

IL-7R precedes KLF2 expression after positive selection. IL-7R and GFP (KLF2) expression in thymocytes from an unmanipulated KLF2-GFP reporter mouse, after gating on DP, or “young,” “middle-aged,” and “old” CD4 SPs 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 numbers owing to the role of IL-7 in survival of thymocytes at the DN stage (11, 19). However, as previously reported, we found 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 thymocytes (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 NKT cells by gating out CD25, γδ TCR, and NK1.1+ cells. Because 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 SPs. Indeed, the CD4 SPs from the Il7ra−/− thymus appeared more mature (HSAlow, Qa2high), consistent with retention, although CD69 and CD62L, which also change with SP maturation, were not as severely affected (Fig. 2B).

FIGURE 2.
FIGURE 2.

KLF2 is expressed in IL-7R KO thymocytes. 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 mouse 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 with wild-type (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-7R signals

Because 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-7R signaling. To allow thymocytes to develop normally through the DP stage and then block the IL-7R, we devised a short-term Ab blockade strategy (Fig. 3A). We limited the blockade to 6 d 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 (26).

FIGURE 3.
FIGURE 3.

IL-7R blockade does not prevent thymocyte emigration. A, Experimental design and dosing schedule for IL-7R 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) (filled histogram) and evaluated 48 h later. B, Analysis of Kb-positive (CD4 and CD8 SP) thymocytes with a secondary Ab (allophycocyanin goat anti-rat IgG). C, Thymocytes from anti–IL-7R–treated mice (bottom panel) or isotype control-treated mice (top panel) were stimulated with 25 ng/ml IL-7 in vitro. After 20 min, intracellular staining was performed with an Ab to phospho-STAT5. Histograms show CD4 SP thymocytes (n = 4 animals/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-negative, CD4 SP gate. H, Ratio of CD4 to CD8 SP in the treated and control mice. Immature CD8 SPs were excluded from the analysis. Control and IL-7R blockade: n = 3.

First, we evaluated whether the in vivo blockade of IL-7R was effective. Using a goat anti-rat secondary Ab, 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). Additionally, the total thymocyte number decreased ∼50% with this short-term IL-7R Ab treatment (Fig. 3D). Taken together, these data indicate that the Ab 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, 3F). 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 d. Again, the S1P1 inhibitor led to an accumulation of labeled cells whereas 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 KO mouse is embryonically lethal from similar endothelial defects as the KLF2 KO mouse (18). Additionally, it has been reported that ERK5 is required for KLF2 expression in mouse embryonic fibroblast cells and that 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 (27).

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. Whereas 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.
FIGURE 4.

ERK5-deficient T cells develop and express KLF2 normally. A, Representative CD4 by CD8 thymocyte profiles (left two panels) and spleen (right two panels) from CD4-cre/Erk5fl/fl and littermate control mice. B, Cell surface phenotype of CD4 SP thymocytes. Gray solid indicates littermate control; black line, CD4-cre/Erk5fl/fl. C, Analysis of ERK5 and KLF2 mRNA from sorted Qa2-expressing, mature CD4 SP thymocytes. Fold change was standardized to hypoxanthine phosphoribosyltransferase and graphed relative to expression in WT mice. Error bar indicates SD. nd, not detected

Discussion

After we generated our data, Arthur’s group (21) published similar findings with ERK5-deficient T cells from conditionally deficient mice. They reported no defect in development for ERK5-deficient T cells and normal numbers of T cells in the spleen and lymph node. Although 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 differences in KLF2 target genes, such as S1P1 and CD62L, were observed. Taken together, ERK5 does not appear to play a critical, nonredundant role in T cell development.

Signaling through the IL-7R, in contrast, has been suggested to play a major role in the survival and function of T cells at multiple steps during development (10, 28). 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 naive T cells is IL-7–independent (29). Taken 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 (28). However, that study employed a gain of function approach to suggest that IL-7 signals can drive CD8 T cell development, and it did not test the hypothesis using loss of function approaches, such as we used in this study. Interestingly, CD8 T cell development did not occur in the absence of all γc cytokine signaling, which includes IL-7 (28). 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.

Note 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 ideas expressed in Park et al. (28). 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, which happens as cells transition from semimature SP to mature SP. It is this final stage where KLF2 is upregulated and which is still largely not understood in genetic/biochemical terms. Because 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 an as yet undefined mechanism.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants AI038903 (to S.C.J.) and AI039560 (to K.A.H.).

  • Abbreviations used in this article:

    DN
    double-negative
    DP
    double-positive
    HSA
    heat-stable Ag
    KLF2
    Krüppel-like factor 2
    KO
    knockout
    SP
    single-positive
    WT
    wild-type.

  • Received July 6, 2010.
  • Accepted November 6, 2010.

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