ERK Signaling Controls Innate-like CD8+ T Cell Differentiation via the ELK4 (SAP-1) and ELK1 Transcription Factors

In mouse thymocyte development, signaling by the TCR through the ERK pathway is required for positive selection of conventional naive T cells. The Ets transcription factor ELK4 (SAP-1), an ERK-regulated cofactor of the SRF transcription factor, plays an important role in positive selection by activating immediate-early genes such as the Egr transcription factor family. The role of ELK4–SRF signaling in development of other T cell types dependent on ERK signaling has been unclear. In this article, we show that ELK4, and its close relative ELK1, act cell autonomously in the thymus to control the generation of innate-like αβ CD8+ T cells with memory-like characteristics. Mice lacking ELK4 and ELK1 develop increased numbers of innate-like αβ CD8+ T cells, which populate the periphery. These cells develop cell autonomously rather than through expansion of PLZF+ thymocytes and concomitantly increased IL-4 signaling. Their development is associated with reduced TCR-mediated activation of ELK4–SRF target genes and can be partially suppressed by overexpression of the ELK4–SRF target gene EGR2. Consistent with this, partial inhibition of ERK signaling in peripheral CD8+T cells promotes the generation of cells with innate-like characteristics. These data establish that low-level ERK signaling through ELK4 (and ELK1) promotes innate-like αβ CD8+ T cell differentiation, tuning conventional versus innate-like development.

D uring development of conventional ab T cells in the thymus, weak TCR signals ensure survival of nonself-reactive thymocytes, whereas strong TCR signaling in self-reactive thymocytes drives their apoptotic elimination (reviewed by Ref. 1,2). ERK signaling downstream of TCR engagement is essential for thymocyte positive selection but not for negative selection (3,4). TCR signaling is also important for development of innate-like ab CD8 + T cells, which express high levels of the Eomes transcription factor and which manifest effector functions immediately upon challenge (5)(6)(7). For example, Itk-null and Slp76 Y145F mutations impair positive selection but increase innate-like ab CD8 + T cell numbers (8)(9)(10)(11). At least in the case of Itk, these phenotypes reflect diminished ERK signaling (8,9), suggesting that weak ERK signaling from lower-affinity TCRs favors innate-like T cell development (reviewed by Ref. 6,7).
The study of innate ab CD8 + T cell development is complicated because it can occur both cell autonomously and in response to cell-extrinsic cues. The latter includes IL-4, which is produced by cells expressing the PLZF transcription factor and influenced by the Itk, Klf2, Id3, Irf4, and Cbp genes, and lymphopenic conditions in the periphery (12, 13; for review, see Ref. 14). Nevertheless, the Nfkb1 and Bcl11 genes contribute cell autonomously to development of innate-like CD8+ T cells, whereas the effects of Itk and Irf4 are at least partly cell autonomous (15)(16)(17). Irf4 is directly induced in response to TCR signaling in an Itk-dependent manner (17), but the relation of Nfkb1 and Bcl11 to TCR signaling remains to be elucidated.
Given the relationship between TCR signal strength and innatelike ab CD8 + T cell development, we set out to evaluate the contribution of ELK4 and ELK1. We demonstrate that ERK signaling to ELK4 and ELK1 acts to limit differentiation of innatelike ab CD8 + T cells in the thymus and periphery, at least in part through expression of the ELK4-SRF target Egr2. Dampening of the ERK signaling upstream of these factors similarly generates peripheral CD8 + T cells with memory-like characteristics. Thus, in response to TCR signals, the ELK4-SRF pathway is central to directing differentiation programs in ab CD8 + T cells. specified in the text), and Egr2Tg and CD4Cre deleter (25). Young adult (6-10 wk) age-and sex-matched animals were used in all experiments. For reconstitution, 8-to-10-wk-old female Rag2 2/2 mice watered on acidified water for 1 wk were irradiated (twice with 500 rad separated by 3-h interval), and bone marrow from the femurs of 6-to-8-wk-old donor mice (0.5-1.0 3 10 6 cells) was injected into the tail vein. Analysis was performed 6 wk later. In some experiments, bone marrow was depleted of T cells and experimental (CD45.2 + ) cells were mixed with (CD45.1 + CD45.2 + ) cells, and 5 3 10 6 cells were injected into lethally irradiated B6 SJL (CD45.1 + ) hosts, having received two 600-rad doses at a 3-h interval. Animals were maintained under specificpathogen-free conditions in the Crick Biological Resources Unit. Animal experimentation was approved by the Crick Animal Ethics committee and carried out under Home Office Procedure Project License 80/2602.

Cell proliferation assays
To assess cell division kinetics, FACS-sorted ab CD8 + T cells were stained with CFSE (5 mM; Molecular Probes) for 15 min in RPMI 1640 without serum, washed in 10% serum RPMI 1640 to stop the reaction, and subsequently plated with anti-CD3 and CD28 or NP68 peptide.

Real-time PCR
Total RNA was extracted from sorted purified ab CD8 + T cells using a GenElute mammalian total RNA kit (Sigma-Aldrich) and subjected to DNase I treatment (Ambion), and cDNA was generated using the Transcriptor cDNA synthetic kit (Roche). Target genes were analyzed using SYBR Green-based real-time PCR (Invitrogen, Carlsbad, CA). Relative template cDNA abundance was calculated using standard curve method, normalizing to Rps16 cDNA, whose level was invariant under all conditions. Primers sequences are available upon request.

Statistical analysis
Data were analyzed with Graph Pad Prism 6. Bar and dot charts are expressed as mean 6 SEM, and data were analyzed using the unpaired and paired parametric t test.
We assessed the functionality of Elk4-null and Elk4 Elk1-null ab CD8 + TCRb hi thymocytes. Upon stimulation with PDBu and ionomycin, purified Elk4-null ab CD8 + SP thymocytes produced substantially increased amounts of IFN-g, a heightened effector function associated with both memory and innate T cells (14, 27, 28) ( Fig. 2A). Furthermore, Elk4-null ab CD8 + thymic T cells exhibited an enhanced sensitivity to TCR signal, as judged by their proliferative response under limiting activation conditions (Fig. 2B). To exclude the possibility that this reflects altered TCR repertoire, we also examined cells expressing a defined TCR. As with polyclonal ab CD8 + thymic T cells, Elk4 Rag2-null F5 transgenic thymic T cells also exhibited enhanced proliferation compared with their WT counterparts upon activation by F5 cognate NP68 peptide (Fig. 2C). Taken together, these observations are consistent with the idea that reduced ELK4-SRF signaling promotes development of ab CD8 + T cells with innate-like characteristics.

Innate-like CD8 + T cells develop in the thymus
Activated and memory peripheral T cells can traffic to specific locations, including the thymus (30,31). The presence of innatelike cells in the thymus could reflect the altered homeostasis of peripheral CD8 T cells migrating back into the thymus because ab CD8 + T cells with memory-like phenotypes are present in Purified ab TCRb hi CD8 + SP thymocytes from Elk4 +/2 and Elk4 2/2 thymus were activated with PDBu and ionomycin and stained for intracellular IFN-g 5 h later. Three animals per genotype; error bars represent SEM. Statistical significance: *p , 0.05 (unpaired t test). (B) Proliferation of CFSE-labeled FACSsorted ab CD8 + SP WT and Elk4 2/2 thymocytes stimulated by CD3/CD28 for 48 h. The percentage of divided cells is shown. Two independent experiments were performed. (C) Proliferation of CFSE-labeled FACS-sorted F5 Rag2 2/2 and F5 Elk4 2/2 Rag2 2/2 CD8 + thymocytes 48 h following stimulation with NP68 peptide and irradiated splenocytes from C57BL6 (APCs). Two independent experiments were performed. peripheral lymphoid organs (see below). To exclude this possibility, we analyzed 1-wk-old animals, in which mature T cells have not yet become established in secondary lymphoid organs. Increased numbers of innate-like cells expressing CD44, CD122, and Eomes were also observed in this setting in Elk4-null animals (Fig. 3A). Moreover, similar results were observed upon E15.5 Elk4-null FTOC (Fig. 3B). Taken together, these results show that Elk4 inactivation acts in the thymus to promote the generation of innate-like ab CD8 + T cells.

Differentiation of Elk4-null innate-like CD8 + T cells is cell autonomous
Innate-like ab CD8 + T cells can develop either cell autonomously (15)(16)(17) or in response to exogenous IL-4 produced by T cell subsets expressing the PLZF transcription factor (12,13). We used a mixed bone marrow chimera approach to address the issue more directly (Fig. 4A). Irradiated CD45.1 WT host animals were reconstituted with WT bone marrow mixed with marrow from WT or Elk4-null animals in either a 4:1 or 1:4 ratio. Six weeks later, the relative contribution and phenotype of the adoptively transferred cells to the thymus population was evaluated, the donor populations being distinguished by their CD45.2 or CD45.1/2 surface markers. WT CD45.1/2 cells exhibited no difference in expression of the CD44, CD122, and HSA memory markers, regardless of whether the original transfer contained excess WT or Elk4-null cells (Fig. 4B, 4D, black versus blue). Similarly, Elk4-null CD45.2 thymocytes exhibited elevated innate-like marker expression regardless of whether they formed the minority or the majority of the transferred population (Fig. 4C, 4D, red versus green, red versus blue). Similar results were obtained in Rag2 2/2 hosts reconstituted with mixed WT and Elk4-null bone marrow (Supplemental Fig. 1).
We next investigated PLZF and CD1d expression in Elk4 Elk1-null thymus. The proportions of thymocytes expressing PLZF or CD1d were not affected, but the absolute number of iNKT cells was increased in line with the increased thymic cellularity observed in Elk4 Elk1-null mutant mice (Fig. 5A, 5D). The intracellular induction levels of IL-4 after PMA/ionomycin stimulation were, however, significantly reduced in Elk4 Elk1-null mice, both in total thymocytes (Fig. 5B) and in PLZF-gated and iNKT-gated thymocytes (Fig. 5C). Among individual PLZF-expressing thymocyte populations, the frequency of iNKT cells was unaffected, although their numbers increased, reflecting increased thymic cellularity (Fig. 5D), but the proportion of CD4 + TCRb hi CD1-d tetramernegative thymocytes was reduced (Fig. 5E). PLZF expression levels in both populations were unaffected (Fig. 5F). Taken together, these data show that generation of Elk4-null innate-like ab CD8 + T cells is substantially cell autonomous and does not reflect alterations in PLZF expression and increased IL-4 expression.
Innate-like ab CD8 + cell development requires immediate-early gene expression Studies in double-positive (DP) thymocytes have shown that TCR signaling to ELK4 and ELK1 activates the classical immediateearly gene set including Egr and AP1 family members (19,20). Quantitative RT-PCR confirmed that both in ab CD8 + TCRb hi thymocytes and peripheral naive ab CD8 + T cells, inactivation of Elk4 led to reduced transcription of classical immediate-early genes at early times following TCR cross-linking (Fig. 6A, 6B). We previously showed that ectopic expression of Egr family members could rescue the positive selection defect in Elk4-null animals (20). To test the role of ELK4-SRF target genes in innatelike T cell development, we used a conditional transgene approach to express EGR2 in DP thymocytes (25). Egr2 expression partially rescued the positive selection defect in Elk4-null animals, reducing the proportion of CD44 hi CD122 hi CD8 + thymocytes and Eomes expression levels (Fig. 6C). These data are consistent with a role for EGR2 and potentially other Egr family members or other ELK4-SRF targets in development of innate-like CD8 + T cells in the thymus.

Innate-like Elk4-null CD8 + T cells accumulate in the periphery
We next investigated whether Elk4 inactivation affected accumulation of innate-like T cells in the periphery. Increased numbers of CD44 hi CD122 hi ab CD8 + T cells, expressing higher levels of Eomes transcripts, were present in the lymph nodes and spleens of Elk4-and Elk4 Elk1-null animals (Fig. 7A). Similar to ab CD8 + SP thymocytes, these cells produced more IFN-g after short stimulation and exhibited enhanced sensitivity in response to TCR signaling, as detected by tritiated thymidine incorporation and CFSE staining (Supplemental Fig. 2). Elk4-null mice are defective in thymocyte positive selection and exhibit lymphopenia (19), which can promote differentiation of memory-like CD8 + T cells in the periphery (14). To exclude this possibility, we analyzed peripheral T cells in animals reconstituted with equal proportions of Elk4-null and WT bone marrow. Again, both lymph nodes and spleen in these animals contained elevated proportions of Elk4-null CD44 hi CD122 hi ab CD8 + T cells that expressed Eomes transcripts at a high level (Fig. 7B) and that produced high levels of IFN-g following stimulation with PDBu and ionomycin (Fig. 7C). Elevated numbers of thymic and peripheral innatelike ab CD8 + T cells were also detected in mixed reconstitutions with Elk4-null cells expressing the MHC class I-specific F5 TCR (Supplemental Fig. 3) and so are unlikely to arise because of altered TCR repertoire in Elk4-null animals. These results suggest that the increased number of peripheral

memory-like ab CD8 + T cells is not a result of lymphopenia in
Elk4-null animals.

Reduced ERK signaling in peripheral T cells promotes development of cell with innate-like properties
We next investigated whether memory-like CD8 T cells could be generated de novo in the periphery. Previous work has shown that weak TCR signals in peripheral CD8 + T cells, generated by interaction with self-peptide/MHC, promote the differentiation into innate-like cells with memory characteristics (reviewed by Ref. 14). Our results suggest a model in which Elk4 inactivation effectively decreases ERK signaling to transcription downstream of the TCR, so we tested whether directly decreasing ERK signaling to ELK4 in WT peripheral cells would increase their differentiation into memory-like cells. We used a system in which naive T cells are subjected to TCR cross-linking in vitro, followed by culture in IL-2; this treatment induces T bet and Eomes expression and causes them to produce IFN-g and TNF immediately upon subsequent TCR restimulation (32). F5 Rag2-null CD8 + T cells were used to study the response of cells expressing a defined TCR.
Inclusion of the MEK inhibitor U0126 during the initial TCR ligation phase progressively limited both ERK activation and Egr1 expression (Fig. 8A). Submaximal concentrations of U0126 reduced T-bet expression at early times of IL-2 culture and increased Eomes expression at late times (Fig. 8B). After prolonged culture in IL-2, U0126-treated F5 ab CD8 + T cells exhibited increased production of IFN-g and granzyme B upon stimulation with PDBu and ionomycin (Fig. 8C). Similar results were obtained when we examined purified naive CD44 lo Elk4-null FIGURE 6. Reduced immediate-early gene expression is associated with innate-like CD8 + T cell development. (A) CD8 SP TCRb hi thymocytes and (B) peripheral CD8 SP CD44 lo CD122 lo cells were activated using aCD3 and aCD28, and expression of Egr1 and Egr2 at the indicated times was analyzed by quantitative RT-PCR. (C) Rag2 2/2 animals were reconstituted either with bone marrow from animals carrying a conditional EGR2 transgene, along with a CD4-Cre transgene to allow expression of EGR2 at the DP stage of thymocyte development, with bone marrow from Elk4 2/2 CD4-Cre, or a combination of these genotypes. TCR b hi CD8 thymocytes were analyzed for CD44, CD122, and intracellular Eomesodermin as in Fig. 1. Error bars represent SEM; n $ 5 animals per group. Statistical significance: ****p , 0.0001 (unpaired t test).
ab CD8 + T cells in this assay (Supplemental Fig. 4A). Experiments with T cells expressing the F5 TCR showed that these changes were also dependent on SRF, the targeting partner protein for ELK4 (data not shown). Together, these data are consistent with a model in which decreased ERK signaling to ELK4 target genes in peripheral or thymic ab CD8 + T cells favors the development of T cells with memory-like characteristics (Fig. 8D).

Discussion
TCR signaling to transcription via the Ets protein ELK4 (SAP-1), its relative ELK1, and their partner transcription factor SRF is critical for thymocyte positive selection (19)(20)(21)(22). In this study, we have shown that ELK4 and ELK1 act redundantly and cell autonomously downstream of ERK signaling to limit the generation of innate-like ab CD8 + T cells with memory characteristics in the thymus. These cells exhibit hallmarks of conventional memory CD8 + T cells, with enhanced expression of the T-box transcription factors Eomes and T-bet and increased proliferation and production of IFN-g in response to TCR ligation (27)(28)(29). The effect of ELK4 inactivation is unlikely to reflect a change in TCR repertoire that preferentially selects innate-like cells because increased innate-like ab CD8 + differentiation occurred in Elk4-null cells expressing the MHC class I-specific F5 TCR. Moreover, innatelike cells were also present in neonatal and embryonic animals, in which the TCR repertoire is limited (33). Like other ELK4 phenotypes, generation of innate-like CD8+ T cells was further enhanced by additional inactivation of Elk1. Transgenic expression of the ELK4 target gene Egr2 reduced the innate-like ab CD8 + T cell development in Elk4-null animals and rescued the positive selection defect. Our results are consistent with the proposal that innate-like differentiation is favored by low TCR signal strength (6, 7) (Fig. 8D).
We used a chimeric reconstitution approach to show that ELK4 acts cell autonomously in innate-like ab CD8 + T cell formation and that this is associated with increased Eomes expression. Innate-like ab CD8 + T cell formation is also controlled cell autonomously by Nfkb1 (16,34) and Bcl11b (15), and Irf4 acts partly cell autonomously (17,35). Previous work has demonstrated that generation of innate-like ab CD8 + T cells in the thymus and periphery can also be induced by cell-extrinsic pathways. Genes, such as Itk, Klf2, Id3, and Cbp, influence innate-like T cell development by increasing levels of IL-4, produced by elevated numbers of PLZF + cells such as iNKT and CD4 + TCRb hi cells (12,13,reviewed by Ref. 14). However, we found that proportions of iNKT cells were unchanged in Elk4 Elk1-null thymocytes, whereas CD4 + TCRb hi cells were decreased, and PZLF expression levels were unchanged in both cell subsets. Moreover, innate-like thymic ab CD8 + T cells in Elk4-null animals expressed markers associated with conventional or lymphopenia-induced memory cells but not IL4-induced memory. IL-4 production in total PLZF + and iNKT + thymocytes was reduced in Elk4 null animals and further reduced in Elk4 Elk1-null thymocytes. Thus, IL4 signaling cannot account for the increased innate-like ab CD8 + thymocyte differentiation that we observe in Elk4-null animals.
Several studies suggest that in addition to promoting positive selection, TCR signaling controls development of innate-like ab CD8 + T cells (reviewed by Ref. 6, 7). Both Itk-null (8,9) and mutant Slp76 Y145F (10, 11) thymocytes exhibit impaired positive selection and increased innate ab CD8 + T cell numbers. Moreover, these phenotypes can be partially suppressed by expression of the hypersensitive ERK sem protein, suggesting they are directly controlled by ERK signaling (8,9). Consistent with this idea, we found that inhibition of ERK signaling promoted the differentiation of cultured peripheral T cells to a memory-like phenotype upon TCR ligation and culture in IL2.
The genomic targets of ELK4-SRF signaling include members of the AP-1 and Egr transcription factor families. We found that transgenic expression of EGR2 suppressed development of innatelike ab CD8 + T cells in Elk4-null thymus, suggesting that EGR2 or other Egr family members act downstream of ELK4; however, it is likely that other ELK4-SRF target genes may be involved because the suppression observed was incomplete. Interestingly, inactivation of Id3, an ERK-responsive Egr target gene (36), also increases innate-like ab CD8 + T cell development but does not do so cell autonomously (12). Moreover, unlike ELK4, Id3 inactivation impairs both positive and negative selection (26). Together, these observations indicate that loss of Id3 expression downstream of ELK4 cannot be responsible for innate ab CD8 + T cell development in Elk4-null animals.
Our data show that ELK4, a major nuclear ERK target in thymocytes, acts downstream of ERK signaling to suppress innate-like ab CD8 + T cell development, acting via target genes such as Egr2. We propose that inactivation of Elk4 attenuates TCR signaling, thereby effectively mimicking low-affinity TCR signaling, leading to increased numbers of innate-like ab CD8 + T cells in addition to impairing positive selection. We note that ITK signaling acts upstream of SRF (37) and that inhibition of ITK signaling potentiates the effect of Irf4 inactivation on innate-like ab CD8 + T cell development (17). This suggests that the ELK4-SRF and IRF4 pathways might cooperate, and, indeed, Irf4 protein levels, but not transcripts, are reduced in Elk4-null thymus (D. Maurice, unpublished observation). It is interesting to note that AP1 [whose components, such as JunB, are Elk4 target genes in T cells (20)] functionally cooperates with IRF4 (38,39).