T Cell Fate and Renders Their Functional Maturation Notch Independent

Immunity Article

Marked Induction of the Helix-Loop-Helix Protein Id3 Promotes the gd T Cell Fate and Renders Their Functional Maturation Notch Independent

Jens Peter Holst Lauritsen,1,4 Gladys W. Wong,2,3,4 Sang-Yun Lee,1,4 Juliette M. Lefebvre,1 Maria Ciofani,2,3 Michele Rhodes,1 Dietmar J. Kappes,1 Juan Carlos Zu´ n˜ iga-Pﬂu¨cker,2,3,* and David L. Wiest1,* 1Blood Cell Development and Cancer Keystone, Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA 2Sunnybrook Research Institute 3Department of Immunology University of Toronto, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada 4These authors contributed equally to this work *Correspondence: [email protected] (J.C.Z.-P.), [email protected] (D.L.W.) DOI 10.1016/j.immuni.2009.07.010

SUMMARY existing data do not easily fit either model, as originally proposed, emerging evidence supports an instructional model in which ab and gd T cells arise from a common thymocyte TCR signal strength dictates fate adoption (Ciofani et al., 2006; progenitor during development in the thymus. Haks et al., 2005; Hayes et al., 2005). Indeed, recent single-cell Emerging evidence suggests that the pre-T cell progenitor analyses defined the CD4ÀCD8ÀCD44ÀCD25+ (DN3) receptor (pre-TCR) and gd T cell receptor (gdTCR) stage as the developmental step at which T-lineage commitment play instructional roles in specifying the ab and gd is completed (Ciofani et al., 2006). These studies further demon- T-lineage fates, respectively. Nevertheless, the strated that ectopic expression of a pre-TCR or gdTCR in Rag2-deficient DN3 thymocytes was capable of instructing signaling pathways differentially engaged to specify commitment to the ab and gd lineages, respectively (Ciofani fate and promote the development of these lineages et al., 2006). Moreover, it was recently shown that the linkage remain poorly understood. Here, we show that differ- between specific TCR expression and fate selection can be ential activation of the extracellular signal-related severed genetically in that a single TCR complex could induce kinase (ERK)-early growth response gene (Egr)- the differentiation into either fate when the resulting signals inhibitor of DNA binding 3 (Id3) pathway plays were modulated (Haks et al., 2005; Hayes et al., 2005). These a defining role in this process. In particular, Id3 findings support the hypothesis that the strength of the TCR expression served to regulate adoption of the gd signal is a critical, lineage-determining factor and that this deter- fate. Moreover, Id3 was both necessary and sufficient mination can be made irrespective of the TCR complex from to enable gd-lineage cells to differentiate indepen- which the signals arise. dently of Notch signaling and become competent Our previous analyses suggested that the stronger signals that promote adoption of the gd-fate involve pronounced activation IFNg-producing effectors. Taken together, these of the extracellular singnal-related kinase (ERK)-early growth findings identify Id3 as a central player that controls response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway both adoption of the gd fate and its maturation in (ERK-Egr-Id3 pathway) (Haks et al., 2005; Hayes et al., 2005). the thymus. Nevertheless, it remained unclear how differential ERK-Egr-Id3 signaling might function in regulating fate choice or how it might INTRODUCTION be integrated with signaling input from other pathways, such as Notch. One attractive target of strong TCR signals is E proteins, T cells comprise two major lineages that arise from a common which are basic helix-loop-helix transcription factors that bind progenitor and are identified by the T cell receptor (TCR) complex DNA at E-box motifs (CANNTG) (Murre, 2005). Mice in which E they express, ab and gd (Ciofani et al., 2006; Dudley et al., 1995). protein genes are ablated (e.g., mice lacking E2A, HEB, or Nevertheless, the molecular processes that underlie specifica- both) exhibit severely perturbed development of ab T cells but tion of the ab and gd fates remain poorly understood. Two models show relatively mild effects on gd T cell differentiation (Bain have been advanced to explain the role of the TCR in this fate et al., 1999; Barndt et al., 2000; Wojciechowski et al., 2007). decision: stochastic and instructional (Hayday and Pennington, Accordingly, strong TCR signals might selectively impair ab 2007). The stochastic model stipulates that fate adoption is T cell development by phenocopying E protein deficiency, either independent of TCR signals, which serve only to rescue survival through marked induction of Id3, which can heterodimerize with of the appropriately matched, predetermined fate. Conversely, E proteins and block their function, or by increased ERK activa- the instructional model posits that TCR signals direct adoption tion, which can induce E protein degradation through a phos- of the fate that matches the TCR from which the signal is derived phorylation-dependent mechanism (Nie et al., 2003; Ordentlich (i.e., pre-TCR for the ab fate and gdTCR for the gd fate). While et al., 1998).

Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. 565 Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 1. Id3 Is Required for Egr1-Mediated Promotion of gd Development (A) Expression levels of Egr1, Egr3, and Id3 mRNA in thymocyte subsets. Egr and Id mRNA expression was quantiﬁed in triplicate in the indicated cell populations by real-time PCR, standardized using b-actin, and normalized to expression in DN3. Results ± SD are depicted graphically. (B and C) Induction of gd development by enforced expression of Egr1 is dependent upon Id3. E13.5 Id3+/À and Id3À/À fetal thymocytes were transduced with empty vector (LZRS) or Egr1 (LZRS- Egr1), seeded into thymic lobes, and placed in fetal thymic organ culture for 2 days. Develop- mental progression was assessed by ﬂow cytometry on electronically gated transduced (GFP+) cells. The absolute number of gd-lineage cells (gdTCR+ or gdTCR+CD24lo) and ab-lineage DP thymocytes per lobe is depicted graphically in (C). Results are representative of three experiments performed.

E proteins may also serve as a common downstream focal findings implicate Id3 as an important molecular-determining point for TCR and Notch signaling. Notch molecules are surface factor in enforcing T-cell-fate decisions, integrating Notch and receptors involved in a wide variety of cell-fate decisions, TCR signals, and enabling functional maturation. including ab and gd lineage commitment (reviewed by Hernan- dez-Hoyos and Alberola-Ila, 2003; Radtke et al., 2004). Recently, RESULTS it was determined that ab-lineage precursors are dependent upon Notch signaling throughout their pre-TCR-induced Id3 Is Required for Egr1-Mediated Regulation of Fate differentiation program to the CD4+ CD8+ DP stage; however, Adoption gd-lineage thymocytes become Notch-independent upon We have previously shown that the ERK-Egr1-Id3 pathway plays expression of the gdTCR complex (Ciofani et al., 2006). Like an important role in the specification of T-lineage fates (Haks TCR signaling, Notch signaling can suppress E protein function et al., 2005). Expression of Egr1, Egr3, and Id3 mRNA was found through ERK activation or Id3 induction (Nie et al., 2003; Orden- to be higher in thymic gd cells than in DN3 and DN4 precursors, tlich et al., 1998; Reynaud-Deonauth et al., 2002). We suggest a representing a mixture of ab and gd lineage progenitors (Fig- model in which the lineage-specific requirement for Notch ure 1A) (Ciofani et al., 2006; Dudley et al., 1995). Moreover, as re- signaling is determined by the relative strength of TCR signals, ported previously, retroviral transduction of Egr1 into C57BL/6 through effects on E protein activity. Accordingly, we hypothe- E13.5 fetal thymocytes markedly increased the frequency and size that Notch-independent differentiation is conferred on number of mature gd T cells (i.e., CD24lo TCRgd+) at the expense gdTCR-expressing cells through strong TCR signals, which are of ab-lineage-committed DP thymocytes (Figures 1B and 1C) capable of repressing E protein activity below the threshold (Haks et al., 2005). The ability of ectopically expressed Egr1 to required for gd differentiation without Notch signals. In contrast, simultaneously promote gd development and oppose the devel- the pre-TCR signals that promote the ab fate are too weak opment of ab lineage cells was dependent on the Egr target, Id3, without the assistance of Notch to suppress E protein activity as these effects are markedly diminished in Id3-deficient cells sufficiently to surmount the E protein mediated checkpoint at (Figures 1B and 1C). Id3 deficiency did not completely eliminate the DN3 stage (Engel et al., 2001; Engel and Murre, 2004). the ability of Egr1 transduction to alter cell fate, perhaps because Because of the ability of Id3 to regulate E protein activity, our of partial compensation by other Egr targets, such as Id2 model suggests that Id3 would be a key molecular mediator (Figure S1 available online). Taken together, these data implicate that facilitates gd-T cell commitment and differentiation. the ERK-Egr-Id3 pathway as a key component of the strong Here, we show that Id3 is required for strong TCR signals to signals that modulate ab-gd lineage fate in the thymus. both promote adoption of the gd fate and oppose the ab-fate outcome. Moreover, although in most cases Id3 is required for Id3 Is Required for Development of the Normal the differentiation of the broader repertoire of non-autoreactive Repertoire of gd T Cells gd-T cells, in certain cases when gd-TCR affinity for ligand was Because Id3-deficiency impaired the Egr-induced development very high, Id3 appears to restrict the differentiation potential of of fetal gd T cells, we asked whether gd cell development that particular TCR-Vg subset. Id3 also plays a critical role in was similarly perturbed in adult mice. Surprisingly, we found conferring Notch-independence to developing gd-lineage cells that Id3 deficiency resulted in an increased number of CD24lo and in arming them to proliferate and produce IFNg in response gd-lineage cells in the thymus (Figure 2A). Consistent with to stimulation. This function of Id3 requires its ability to interact a recent report, the expansion appeared to be restricted to with and suppress E protein targets. Taken together, these a subpopulation of Vg1.1+ gd T cells (Ueda-Hayakawa et al.,

566 Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 2. Id3 Deficiency Disrupts the Devel- opment of gd T Cells (A and B) Id3 deficiency results in expansion of the Vg1.1+ subset. Id3À/À mice were backcrossed to the C57BL/6 background for three generations before tissues from mice of the indicated genotypes were analyzed by flow cytometry. Results are depicted as histograms and summarized graphically on the right. Error bars represent SD. (C and D) Id3 deficiency rescues deletion of autoreactive gd precursors. Thymocyte preparations were produced from Rag2+/À KN6 Tg mice expressing positively selecting ligand (T-10d)or higher-affinity negatively selecting ligand (T-10d/b) and from KN6 Tg mice expressing high-affinity ligand but lacking Id3. Preparations were analyzed by flow cytometry and gated DN cells were displayed as histograms as above. Differences in absolute number of the indicated subsets that are marked by asterisks are statistically significant (p < 0.05). Results are representative of three experiments performed with a minimum of two mice per genotype in each experiment. Error bars represent SD.

Regulation of Lineage Fate by the Egr-Id3 Pathway Does Not Require Alterations in TCR Repertoire The selective inhibition of particular Vg subsets by Id3 deficiency could result from effects on the processes of lineage commitment, selection and maturation, or gene rearrangement (Ueda-Hayakawa et al., 2009). Consistent with the latter possibility, the E protein targets, whose 2009), as other gd cells, including the Vg2 and Vg3 dendritic functions are antagonized by Id3, have been shown to be epidermal T cell (DETC) subsets, were reduced (Figures 2A involved in regulating TCR gene rearrangement (Agata et al., and 2B). Unlike the conventional Vg2 and Vg3 subsets, Vg1.1+ 2007; Bain et al., 1999). Accordingly, it was possible that ectopic gd cells have been suggested to represent so-called NK gd expression of Egr1 and Id3 deficiency were modulating lineage T cells, which are thought to be autoreactive and selected on fate indirectly by altering the TCR repertoire. To test this possi- high-affinity ligands (Felices et al., 2009; Lees et al., 2001; Qi bility, we revisited the role of superinduction of Egr1 and Id3 on et al., 2009). We reasoned that such cells might normally be T cell fate in a model with a fixed TCR repertoire. For this deleted by excessively strong signals but are allowed to survive purpose, we employed KN6 gdTCR Tg Rag2-deficient mice in and expand in the absence of Id3. Since the putative selecting which lineage choice can be manipulated by modulating access ligand(s) for the Vg1.1+ subset have not been identified, we to ligand or signaling output from a single TCR complex (Haks sought to test this possibility by using the KN6 gd TCR transgenic et al., 2005; Pereira et al., 1992). Thymocytes from KN6 Tg (Tg) mice in which KN6 gdTCR Tg thymocytes are positively mice expressing T10d ligand adopt the gd fate (CD4À CD8À selected in the thymus on the nonclassical MHC class Ib mole- gdTCR+ CD24lo), whereas those lacking ligand (i.e., b2M defi- cule, T10d, but are deleted by the 10-fold higher-affinity T-10b cient) commit to the ab lineage, undergo proliferative expansion, (and/or T-22b) ligand (Figure 2C) (Adams et al., 2008; Pereira and develop into DP thymocytes (Figure S2). Accordingly, we et al., 1992). Our hypothesis predicts that deletion of KN6 Tg employed the KN6 Tg model to assess the ability of Egr1 and thymocytes by T-10b ligand should be prevented by Id3 defi- Id3 to modulate lineage fate independently of alterations in the ciency. Indeed, Id3 deficiency blocked deletion of KN6 Tg TCR repertoire. To determine whether ectopic expression of thymocytes by T-10b ligand, resulting in a marked expansion Egr1 would divert cells from the ab lineage to the gd fate, we of KN6 Tg thymocytes with a mature CD24lo phenotype (Fig- ectopically expressed Egr1 in KN6 Tg mice lacking ligand (i.e., ure 2D). These data suggest an apparent dichotomy of Id3 func- committing to the ab lineage; Figure 3A, left panel) (Miyazaki, tion, with Id3 restricting the development of TCR-Vg subsets 1997). Indeed, we found that the Egr1 Tg both antagonized ab- bearing high-affinity gdTCRs while at the same time being lineage development, as indicated by the reduced number of required for development of the broader repertoire of non-autor- DP thymocytes, and promoted development of mature CD24lo eactive gd-T cells. gd-lineage cells (Figure 3A). We next investigated whether the

Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. 567 Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 3. Egr1 and Id3 Can Modulate Lineage Choice without Affecting the TCR Repertoire (A) Enforced expression of Egr1 diverts KN6 Tg thymocytes to the gd fate. Thymocytes from mice of the indicated genotypes were analyzed by flow cytometry: (1) LigÀ, KN6 Tg Rag2À/ÀB2mÀ/À; (2) LigÀEgr1Tg, KN6 Tg Rag2À/ÀB2mÀ/ÀEgr1Tg. (B) The strong TCR signals that promote adoption of the gd fate require Id3. Mice of the indicated genotypes were analyzed by flow cytometry: (1) Lig+, KN6 Tg Rag2À/À; (2) Lig+Id3À/À, KN6 Tg Rag2À/ÀId3À/À. (A and B) Results are depicted as histograms and summarized graphically on the right with each symbol corresponding to an individual mouse. Differences between the absolute number of DP and CD24lo DN thymocytes between these mice are statistically significant (asterisk indicates p < 0.05).

Egr1 target, Id3, was required for strong signals (generated by the context of strong signals, normally suppresses their growth. gdTCR-ligand interactions) to promote gd development and Taken together, these findings indicate that following strong simultaneously oppose development of ab-lineage cells. In TCR signals, Id3 functions to promote the production of CD24lo agreement, Id3 deficiency interfered with the ligand-induced gd-lineage cells and opposes the development of ab lineage, development of mature CD24lo gd-lineage cells while increasing and this is associated with effects on growth and survival. the number of ab-lineage DP thymocytes (Figure 3B). Impor- To gain insight into the molecular basis for the differences in tantly, Id3 deficiency caused proportional changes in ab-lineage survival and proliferation observed in Id3-deficient mice, we DP and CD24lo gd-lineage cells, consistent with an effect on examined the expression levels of molecular effectors of these lineage commitment. These data suggest that the Egr-Id3 pathways. An attractive set of candidates was the RORgt-BclXL pathway plays a critical role in lineage assignment that does axis, shown recently to require the E protein targets of Id3 for not require alterations in the TCR repertoire. Id1 deficiency had expression (Xi et al., 2006). In accord with this model, expression À/À no effect on lineage assignment in this model (data not shown). of RORgt and the prosurvival factor BclXL was elevated in Id3 Notably, the ability of strong signals to promote the gd lineage ISP cells relative to that in Id3+/+ ISP cells, consistent with their was associated with markedly reduced expression of E proteins increased survival (Figure 4C). Interestingly, expression of À/À lo (E47) relative to that observed in cells committing to the ab fate in RORgt and BclXL was not decreased in Id3 CD24 gd lineage the absence of ligand (Figure S3). and, thus, did not correlate with the increased apoptosis of these cells (Figure 4C). However, Bcl-2 expression was substantially Effects of Id3 Deficiency on Fate Adoption Are reduced in Id3À/À CD24lo gd-lineage cells, consistent with their Associated with Changes in Survival and Proliferation increased apoptosis (Figure 4C). These data suggest that Id3 We next wished to determine how strong signals employ Id3 induction in the context of a strong signal is associated with to both promote development of gd-lineage cells and oppose the increased survival of gd-lineage cells and decreased survival ab-lineage fate. Previous studies indicated that the E proteins of ab-lineage cells. Further, these fates correlate with differential, can control cell growth and survival (Engel et al., 2001; Engel cell-context-dependent effects on the expression of the prosur- and Murre, 2004; Xi et al., 2006). Accordingly, we reasoned vival factors Bcl-2 and BclXL, respectively. Expression patterns that repression of E proteins by Id3 might differentially influence of other E protein targets, CDK6 and Bim, were not consistent growth and survival of developing ab and gd T-lineage cells. with alterations in growth and survival (Figure S4). Interestingly, the impaired generation of mature CD24lo gd- Because Id3 deficiency is associated with changes in growth lineage cells in ligand-expressing Id3À/À KN6 Tg mice was asso- and survival of ab and gd lineage cells occurring under the influ- ciated with increased Annexin V staining, suggesting that Id3 is ence of strong TCR signals, we thought it possible that Id3 may required to promote the survival of these cells (Figure 4A). The regulate lineage selection prior to TCR signaling by acting on CD24lo population also exhibited increased BrdU incorporation. precommitted progenitors. Lineage fate has been shown to be Perhaps this contributes to the increased apoptosis among complete in DN3 progenitors and can be controlled by ectopic these cells, since cycling cells are more sensitive to apoptotic expression of TCR complexes in Rag2À/À DN3 thymocytes (Cio- stimuli (Komarova et al., 1997). Effects on survival and prolifera- fani et al., 2006). To determine whether Id3 induction might be tion also appear to be associated with the ability of Id3 to oppose functioning by selectively inducing apoptosis among precom- ab-lineage development (Figure 4B). Indeed, Id3-deficiency mitted ab-lineage precursors, we coexpressed Id3 (to mimic caused an increase in survival among cells that differentiate to a strong signal) with TCRb (pre-TCR-induced ab-fate) in the ab-lineage DP stage (i.e., through a CD8 immature single Rag2À/À DN3 thymocytes. Our results showed that Id3 transduc- positive or ISP intermediate) and accumulate in Id3À/À mice- tion did not result in selective loss of ab-lineage precursors, expressing ligand (Figure 4B). Moreover, Id3 deficiency suggesting that strong signals through Id3 are acting on the increased the fraction of BrdU+ ISP cells, suggesting that Id3, in specification or commitment process to promote gd-lineage

568 Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 4. Id3 Promotes the gd Fate and Opposes the ab Fate, and These Effects Are Associated with Alternations in Growth and Survival (A and B) Id3 promotes survival of mature CD24lo gd-lineage cells and impairs growth and survival of CD8ISP and ab lineage DP. Apoptosis and proliferation of the indicated populations were evaluated flow cytometrically by Annexin staining and BrdU incorporation, respectively. Each symbol represents an individual experiment involving at least three animals, with a line con- necting the animals with a different genotype con- tained within a single experiment (upper panels). The difference in percent (%) of apoptotic cells between Lig+ and Lig+Id3À/À populations was significant (p < 0.05) for the following populations: (1) gdTCR+CD24lo; (2) ISP; and (3) DP. The mean percent BrdU incorporation ± SD is presented graphically (bottom panels) with a minimum of ten mice per condition. *p < 0.05; **p < 0.01. (C) Effects of Id3 on growth and survival are associated with differential regulation of Bcl2 family members. Expression of the indicated genes was measured by real-time PCR on thymocyte subsets purified by flow cytometry. Expression levels were standardized using b-actin and normalized to levels in Rag2À/À DN3 thymocytes. Data are representative of two different experiments. Error bars represent SD. differentiation and oppose the ab fate (Figure S5). Conversely, to Recent reports have suggested that full functional maturation determine whether strong signals promote the generation of of gd-lineage cells requires trans presentation of lymphotoxin-b CD24lo gd-lineage cells by preferentially expanding a small pre- (LTb) to developing gd-lineage cells by DP thymocytes (Penning- committed gd-lineage population, we ectopically expressed the ton et al., 2003; Silva-Santos et al., 2005). This induces a set of KN6 gdTCR in Rag2À/À thymocytes, which lack precommitted genes termed the ‘‘gd-biased gene profile,’’ whose expression gd-lineage cells, and assessed effects on fate adoption in the is enriched in gd cells and linked to function: ICER, Nurr1, presence and absence of ligand (Figure S6). Importantly, KN6 Nor1, Nurr77, and Rgs1 (Pennington et al., 2003; Silva-Santos gdTCR-expressing precursors adopted the gd-fate in the pres- et al., 2005). Because ligand-selected KN6-Tg gd-lineage cells ence of ligand (at the expense of the ab-fate) and, conversely, are functional (Figures 5A–5C), yet develop in the near absence were diverted to the ab fate (at the expense of the gd fate) of DP thymocytes (Figure 3B), we examined whether these cells when ligand expression was suppressed by shRNA (Figure S6). express the gd-biased gene profile. Surprisingly, KN6 Tg gd cells Altogether, these data suggest that strong signals and Id3 are from Id3-sufficient, ligand-expressing mice exhibited increased able to influence lineage commitment per se. The effects on expression for all of the gd-biased genes examined (Figure 5D), ISP and DP survival that we observed in vivo likely occur in indicating that DP-mediated trans conditioning may not be a minority of cells that escape ligand engagement early in devel- absolutely required for their induction. These findings support opment. Recent observations suggest that ligand engagement the notion that elaboration of the gd-biased gene profile is asso- of uncommitted DN thymocytes induces commitment to the gd ciated with gd function (Pennington et al., 2003; Silva-Santos lineage; however, cells that escape ligand engagement and et al., 2005); however, Id3 deficiency appeared to sever this commit to the ab fate appear to become susceptible to deletion linkage. Indeed, despite the fact that proliferation and IFNg by ligand as they differentiate to the ISP or DP stages (Kreslavsky production in response to TCR engagement were impaired in et al., 2008). gdTCR+ cells from Id3-deficient mice, these cells displayed elevated expression of a subset of the gd-biased profile-genes gd-Biased Profile Gene Rgs1 Is Closely Linked (Figure 5D), with the notable exception of Rgs1 expression, with gd T Cell Function which was reduced in these cells. One potential explanation Because Id3 deficiency interfered with gd cell maturation in for this observation is that particular members of the gd-biased response to strong TCR signals, we investigated whether effector profile may be more closely linked to gd-function than others. function was similarly compromised. Id3 deficiency impaired Specifically, Rgs1 expression correlated well with gd cell func- both TCR-induced proliferation and IFNg production (Figures tion in that it was highly induced in functional KN6 Lig+ gd cells 5A–5C). Additionally, this effect was largely restricted to gd- and reduced in those lacking Id3 (Figure 5D); expression of the lineage cells because the TCR-induced proliferation of ab-lineage Nr4a family members (Nurr1, Nor1, and Nur77) was not. The CD4 T cells was not affected by Id3-deficiency, although prolifer- elevated expression of the Nr4a genes in the nonfunctional, ation of Id3À/À CD8+ T cells was somewhat delayed following TCR Id3-deficient gdTCR+ CD24lo cells may relate to their role in activation (Figure S7)(Pan et al., 1999; Rivera et al., 2000). Of regulating cell death in this misdirected population exhibiting note, both proliferation and IFNg production occur only in mature increased apoptosis (Figure 4)(Cheng et al., 1997; Liu et al., gd-lineage cells that are CD24lo (Figure S8)(Pereira et al., 1992). 1994). Our data indicate that induction of some genes within

Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. 569 Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 5. Induction of Id3 by Strong Signals during gd Development Is Required for Functional Competence (A–C) Id3 deficiency impairs TCR-dependent proliferation and production of IFNg. DN thymocytes from KN6+Lig+ and KN6+Lig+Id3À/À mice were labeled with CFSE, stimulated for 48 hr on plate- bound anti-CD3 (10 mg/ml), and analyzed by FACS (A). DN thymocytes from the same mice as in (A) were stimulated as above, and cellular expansion was evaluated on triplicate wells by MTT assay. (B). DN thymocytes were stimulated with plate-bound anti-CD3 Ab for 24 hr, following which the proportion of IFNg producing cells was determined by intracellular staining and flow cytometry. (B and C) Mean ± SD is presented graphically. (D) Expression of the gd-biased gene profile and functional competence can be separated. Expres- sion of the indicated genes was measured on flow cytometrically isolated cell populations by real- time PCR as in Figure 4. Mean ± SD is presented graphically. the gd-biased profile, such as Rgs1, is tightly linked to gd T cell that the ERK-Notch-mediated degradation of E proteins may function, whereas others appear to be separable. be critically important for reducing E protein activity at the b-selection checkpoint (Engel et al., 2001; Engel and Murre, Id3 Induction by Strong Signals Is Necessary and 2004; Huang et al., 2004). This finding provides a potential expla- Sufficient to Promote Notch-Independent Maturation nation for why the generation of ab-lineage DP cells is clearly The above findings provide support for Id3 as an important dependent upon Notch, yet independent from Id3 (Figures 6A molecular effector of the strong signals that dictate gd fate and 6B) (Huang et al., 2004; Nie et al., 2003; Ordentlich et al., choice and maturation. However, it remained unclear whether 1998). Alternatively, Rag2-deficient DN3 cells transduced to other aspects of gd-lineage development might rely on Id3. We express the KN6 gdTCR showed a strong reduction in E47 levels wished to determine whether Id3 induction in the context of irrespective of Notch signals (Figure 6B, right side). Because strong signals was required to confer Notch-independent matu- these data suggest that Id3 plays an important role in conferring ration upon gdTCR-expressing cells (Ciofani et al., 2006). To test Notch independence to gd-lineage cells, we next examined this possibility, we ectopically expressed either a pre-TCR or whether Id3 induction was sufficient. In fact, ectopic expression gdTCR in Rag2À/À DN3 cells and then assessed the role of Id3 of Id3 in Rag2, Id3-double-deficient DN3 cells led to increased in determining the Notch-dependence of their development cellular expansion (Figure 6C) and maturation (e.g., CD24 down- in vitro (Figure 6A). As reported previously (Ciofani et al., 2006), modulation; Figure 6D and Table S1), which were both indepen- pre-TCR (TCRb-transduced) expressing Rag2-deficient DN3 dent of Notch signaling. Moreover, the ability of Id3 to confer cells proliferated robustly and developed to the DP stage (Fig- these capabilities on Rag2, Id3-double-deficient DN3 cells was ure 6A). This differentiation was independent of Id3, as it was abrogated by a mutation that eliminated the ability of Id3 to not impaired by Id3-deficiency but was dependent upon Notch interact with and suppress E protein function (S / P; Figures signaling (i.e., required a Delta-Notch ligand, DL1; Figure 6A). 6C and 6D) (Deed et al., 1997). Id3 expression following trans- Even the ab-lineage DP cells that developed in the gdTCR- duction was approximately 10-fold higher than is expressed expressing cultures were dependent upon Notch signals endogenously by CD24lo gd cells (Figure S8). These results (Figure 6A) (Ciofani et al., 2006; Garbe et al., 2006). In contrast, show that different mechanisms are responsible for mediating gd-lineage commitment and development into mature DN the reduction in E protein activity that is necessary for DN3 cells CD24lo gd cells was not dependent upon Notch signals but to differentiate into either gd or ab lineage cells, with the gd was dependent upon Id3. Indeed, Id3 deficiency markedly option relying solely on Id3 induction, while the ab option reduced the numbers of CD24lo gd-lineage cells, instead divert- requires additional input from Notch signals. ing the gdTCR-expressing cells to the ab-lineage DP stage (Fig- ure 6A), as we observed in vivo (Figure 3). These data indicate Id3 Induction Is Necessary and Sufficient to Promote that Id3 function is particularly important for elaboration of the Functional Competence of gd Lineage Cells gd lineage, but is dispensable for adoption of the ab fate. A recent report implicated ligand ‘‘experience’’ in the thymus as Interestingly, expression of the E protein target of Id3, E47, an important determinant of effector function, arming such gd was reduced by Notch signaling, even in the absence of TCR cells as IFNg-producing effectors (Jensen et al., 2008). Since (preTCR or gdTCR) expression (Figure 6B). However, consistent we have found that ligand-mediated induction of Id3 was neces- with the requirement of Notch signals for differentiation of pre- sary to arm KN6 Tg gd cells for TCR-induced proliferation and TCR-expressing Rag2-deficient DN3 cells, we observed that IFNg production (Figure 5C), we asked whether Id3 was suffi- E47 levels were dramatically reduced only when both pre-TCR cient to promote the functional maturation of Rag2-deficient and Notch signals are present (Figure 6B, left side), indicating DN3 cells. Indeed, ectopic expression of Id3 in Rag2,

570 Immunity 31, 565–575, October 16, 2009 ª2009 Elsevier Inc. Immunity Id3 Controls gd Cell Speciﬁcation and Development

Figure 6. Id3 Induction by Strong Signals Is Necessary and Sufficient to Confer Notch Independence on gd-Lineage Cells (A) Notch independence of gd-lineage cells is impaired by Id3 deficiency. Rag2À/À or Rag2À/À Id3À/À DN3 cells were transduced as indicated and cultured on OP9-DL1 or OP9-Cntl cells for 5 days, and the expression of CD4, CD8, and CD24 was analyzed by flow cytometry. Bar graphs on the right show the fold increases (relative to control transduced cultures) in absolute numbers of DP or gd+ CD24lo DN cells from the indicated transductions, culture conditions, and genotype. (B) Notch signals reduce E protein expression. Equal quantities of protein from detergent extracts of cells cultured as in (A) were immunoblotted with the indicated Ab. (C and D) Ectopic expression of Id3 is sufficient to confer Notch independence on TCRÀ thymocytes. Rag2À/ÀId3À/À DN3 cells were transduced as indicated, cultured on OP9-DL1 or OP9-Cntl cells for 6 days, with the fold increases in cellularity over input shown in (C) and the expression of CD24 and forward-light scatter (FSC) analysis by flow cytometry are shown in (D). These data are representative of at least three independent experiments. Error bars represent SD.

gd-lineage fate and for arming gd T cells to produce IFNg in response to ligand experience.

DISCUSSION

The present report not only provides new insights into the role of Id3 in regulating T-lineage fate assignment in response to strong signals, but also into the role of Id3 in rendering gd-lineage cells inde- Id3-double-deficient DN3 cells enabled these cells to become pendent of Notch signals and functionally competent. Id3 is responsive to stimulation. In response to mitogenic stimulation, required for strong signals to promote adoption of the gd lineage Id3-transduced cells both increased in size (Figure 7A) and and oppose adoption of the ab fate. We recently reported that produced IFNg at amounts comparable to mature CD24lo gd gd-lineage cells are far less dependent on Notch signaling than cells (Figure 7B). Furthermore, Id3 transduction conferred these ab-lineage cells (Ciofani et al., 2006). Based on our present capabilities to Rag2, Id3-double-deficient precursors in the findings, it is now clear that Id3 is both necessary and sufficient absence of Notch receptor-ligand interactions (Figure 7B). As for Notch independence of gd-lineage cells and that this above, the ability of Id3 to promote Notch-independent acquisi- independence requires a functional helix through which Id3 het- tion of effector function was lost when the Id3 mutant construct erodimerizes with, and suppresses the function of, E proteins. was used (Figures 7A and 7B). The Id3-induced acquisition of Finally, Id3 is also necessary and sufficient to arm gd-lineage effector function was accompanied by the induction of ICER cells for effector function defined by TCR- or mitogen-induced and Rgs1, a gd-biased gene profile member that we have found proliferation and IFNg production. These findings place Id3 as to be closely linked to gd function (Figure 7C), but not induction of a central molecular mediator of the strong signals that influence Nurr1, whose expression appears to be separable from gd cell T cell fate, as well as their developmental and functional charac- function (Figure 5). Sox13 is another recently described marker teristics. of gd lineage cells (Melichar et al., 2007); however, while Sox13 It has been suggested for some time that ab- and gd-lineage expression was not detected in DP cells, it was also not found precursors are differentially dependent upon Notch signaling, to be elevated in CD24lo gd-lineage cells, in agreement with though this has been controversial until recently (Garcia-Peydro recent evidence suggesting it may not reliably mark all gd cells et al., 2003; Hernandez-Hoyos and Alberola-Ila, 2003; Radtke (Kreslavsky et al., 2008). Taken together, these data suggest et al., 2004; Washburn et al., 1997). Indeed, we showed that that Id3 is both necessary and sufficient for adopting the while ab-lineage precursors remain Notch dependent during

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Figure 7. Ectopic Expression of Id3 Is Able to Confer Functional Competence on TCRÀ Thymocytes (A) Id3 transduced cells increase in size upon PMA + ionomycin stimulation. (B) Id3 transduction confers competence to produce IFNg on Rag2, Id3-double-deficient DN3 cells. (A and B) Sorted CD45+ GFP+ cells from transduced DN3 cells (as indicated) were stimulated for 36 hr with PMA and Ionomycin (PMA + Iono) or placed in culture without stimulation (Unstim) and (A) analyzed by flow cytometry to determine cell size by forward-light scatter (FSC). (B) Levels of IFNg in culture supernatants were quantified by an antibody-capture ELISA. (C) Id3 transduction confers expression of some gd-biased genes. RNA transcript expression of Rgs1, ICER, Nurr1, and Sox13 was measured by real time PCR as in Figure 5 from sorted CD45+ GFP+ cells from transduced DN3 cells cultured on OP9-Cntl or OP9-DL1 cells as indicated. Error bars represent SD.

et al., 2001). Accordingly, pre-TCR signals may suppress E protein activity, in part by induction of Id3 but are unable by themselves to suppress E protein activity beyond the threshold required for ab-lineage development and, thus, require Notch-ligand interactions to do so. Notch signaling has been reported to suppress E protein function both by ERK-dependent induction of E protein their pre-TCR induced transition to the DP stage, gd precursors degradation (Nie et al., 2003; Ordentlich et al., 1998) and through become independent of Notch signaling upon expression of the induction of Id3, which can directly repress E protein function gdTCR (Ciofani et al., 2006). Nevertheless, it was unclear how (Reynaud-Deonauth et al., 2002). Unlike the weak signals that gdTCR signaling permitted Notch-independent differentiation promote ab-lineage development, the strong signals that confer of gdTCR-expressing DN cells. We now show that Id3 is an Notch-independent differentiation upon gd-lineage cells are important molecular effector of this process. The molecular dependent upon Id3 induction and are sufficient to suppress E basis for this effect has not been established; however, recent protein activity beyond the threshold required for gd-lineage evidence from the Murre lab (as well as genetic analysis from commitment and development without assistance from Notch Drosophila) suggests interplay between E proteins and the Notch activity. pathway (Ikawa et al., 2006; Schwartz et al., 2006). Moreover, Because effects on growth and survival are associated with the numerous reports support the notion that suppression of regulation of lineage fate by Id3 in the context of strong TCR E protein function (at least transiently) is required for early thymo- signals, it is possible that Id3 is not affecting lineage fate per se, cyte differentiation (Engel and Murre, 2004; Koltsova et al., 2007; but rather is affecting the growth and survival of precommitted Murre, 2005). We now propose a model whereby lineage ab or gd lineage progenitors. Nevertheless, a number of lines of commitment and Notch dependence are determined by graded evidence argue against this perspective and for a role of Id3 in reductions in E protein activity mediated by differences in TCR influencing lineage commitment. If strong signals were simply signal strength (Figure S10). DN3 thymocytes are prevented eliminating ab-lineage cells, then Id3 transduction would be pre- from further development by E protein-mediated enforcement dicted to preferentially eliminate pre-TCR-expressing ab-lineage of the b-selection checkpoint, as evidenced by the ability of progenitors; however, Id3 transduction does not do so. E2A deficiency to enable pre-TCR-deficient thymocytes to Regarding the promotion of the gd fate by strong signals (or Egr traverse the b-selection checkpoint and differentiate to the DP transduction), the converse argument could be made that strong stage (Engel and Murre, 2004; Wojciechowski et al., 2007). Para- signals promote the generation of CD24lo gd-lineage cells by pref- doxically, E protein deficiency blocks the development of erentially expanding a small precommitted population. However, pre-TCR-expressing cells beyond the b-selection checkpoint we demonstrated that ectopic expression of the KN6 gdTCR in to the DP stage, suggesting that the induction of ab-lineage Rag2À/À thymocytes (which lack pre-committed gd-lineage cells) development by pre-TCR signals is dependent upon partial or promoted the gd fate and opposed the ab fate in the presence of temporally restricted suppression of E protein activity (Engel ligand and produced the converse effect in the absence of ligand

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(Figure S6). It should be noted that this represents the first that Id3 deficiency separated gd function from expression of example where the expression of a specific gdTCR ligand was the gd-biased gene profile in that the Id3-deficient gd cells demonstrated to be required for selection and development of expressed a subset of the profile genes, with the notable excep- gd-lineage precursors. Altogether, these data suggest that strong tion of Rgs1, and yet were not functional. One possible explana- signals and Id3 drive lineage commitment. tion is that only a subset of the profile genes is uniquely associ- While we had previously reported impaired development of gd ated with gd function. For example, although the function of T cells in fetal Id3-deficient mice, the role of Id3 as a critical Rgs1 in T cells is unknown, our data suggest that Rgs1 expres- molecular effector in gd cell specification and development is sion is tightly correlated with gd T cell function, raising the inter- also revealed by the perturbation of gd development in adult esting possibility that Rgs1 plays an active role in promoting the Id3-deficient mice. A recent report suggested that Id3 functions functional competence of gd T cells. to restrain the development of gd-lineage cells, as gd T cell The role of Id3 in promoting functional competence also numbers were increased in adult Id3-deficient mice (Ueda- provides an important mechanistic insight into a recent report Hayakawa et al., 2009). The expansion was accompanied by regarding the role of ligand in shaping gd T cell effector function. enhanced V(D)J recombination at the TCR loci (g, d, and b) and The report contends that ligand-mediated selection in the thymus an outgrowth of the Vg1.1+ gd cell subset. We observed similar does not play a significant role in shaping the gd TCR repertoire effects of Id3 deficiency, but propose an alternative interpreta- but does influence the nature of effector function (Jensen et al., tion. We suggest that Id3-serves to terminate V(D)J recombina- 2008). That is, gd T cells that purportedly did not encounter a tion following TCR expression (either pre-TCR or gdTCR selecting ligand during development (antigen-naive) are pro- expression), thereby limiting the developmental time window grammed to produce IL-17, whereas antigen-experienced gd during which TCR rearrangement can occur and explaining why T cells produce IFNg. While ruling out the possibility of ligand- TCR rearrangements were more extensive in Id3-deficient mediated selection in the thymus is extremely difficult, we would mice. We further demonstrated that while gd T cell numbers agree that ligand-mediated selection arms gd T cells to produce were increased in Id3-deficient mice, the expansion appeared IFNg. Indeed, our data supports the notion that the induction of to be restricted to the Vg1.1+ subset, as the Vg2 and Vg3 gd Id3 following TCR-ligand engagement plays a critical role. subsets were severely reduced. We think these differential Taken together, our findings place Id3 as a central molecular effects on particular Vg subsets relate to their respective affinities mediator of the strong signals that influence T cell fate, enable for selecting ligands, with Id3-deficiency impairing selection of developing gd T cells to gain Notch-independent maturation, those with intermediate affinity, while allowing those of high and shape their functional attributes. Although it is likely that affinity to escape deletion. This interpretation is supported by Id3 (perhaps with assistance from Id2) mediates these effects our demonstration that Id3 deficiency impairs selection of KN6 by suppressing the function of E proteins, this interpretation gdTCR Tg thymocytes on the intermediate affinity T-10d ligand, remains to be fully characterized. Future efforts will be directed while rescuing them from deletion by T-10b and T22b, for which at determining how the ERK-Egr-Id3 pathway, as well as other their affinity is 10-fold higher (Adams et al., 2008). Indeed, at least mediators of strong signals, is differentially employed to specify some Vg1.1+ gd cells, which are expanded in Id3À/À mice, belong T-lineage fates in the thymus. to an NK gd T cell lineage thought to be selected on high-affinity ligand (Felices et al., 2009; Lees et al., 2001; Qi et al., 2009). Taken EXPERIMENTAL PROCEDURES together, these findings suggest an apparent dichotomy of Id3 function, restricting production of gd cells whose TCR affinity Mice for ligand is very high, while promoting differentiation of the All mice were maintained in the fully accredited animal facilities at either the broader repertoire of nonautoreactive gd T cells. Fox Chase Cancer Center or the Sunnybrook Research Institute, and their use was approved by the Institutional Animal Care and Use Committee. All Along with its role in promoting the Notch independence of mouse strains used were described previously (Haks et al., 2005). Id3À/À developing gd T cells, Id3 plays an important role in the acquisi- mice were kindly provided by Drs. R. Benezra (Memorial Sloan-Kettering tion of effector function by gd lineage cells. We found that Id3 defi- Cancer Center) and Y. Zhuang (Duke University) (Pan et al., 1999) and used ciency impairs the ability of KN6 gdTCR Tg+ cells to proliferate both on the 129 background and after backcrossing to C57BL/6 for three and produce IFNg in response to TCR ligation. Conversely, generations. Egr1Tg mice were provided by Toru Miyazaki (University of ectopic expression of Id3 in the absence of TCR expression Tokyo) (Miyazaki, 1997). conferred the ability to proliferate and produce IFNg in response to mitogenic signals. The Hayday laboratory has reported that Retroviral Transduction and Culture developing gd T cells require presentation of LTb in trans by DP LZRS-pBMN-linker-IRES-eGFP (LZRS) and LZRS-Egr1 vectors have been previously described (Carleton et al., 2002). Murine Id3 was cloned by PCR thymocytes for both functional competence and the expression using primers that spanned the start and stop codons. Serine 49 of Id3 was of a number of genes termed the ‘‘gd-biased gene profile’’ mutated to P (S49 / P) by standard methodology and, following sequence (Pennington et al., 2003; Silva-Santos et al., 2005). DP thymo- verification, was subcloned into pMiY using standard methodology. Viral cytes are not present in appreciable numbers in either the supernatants were produced as described and used to spin-infect progenitors ligand-expressing KN6 gdTCR Tg mice or among Id3-transduced prior to seeding fetal thymic lobes (Il2rgÀ/ÀRag2À/À) or plating on OP9 stromal DN3 cells cultured on OP9-monolayers in vitro. Nevertheless, layers as described (Ciofani et al., 2006; Haks et al., 2003). The effect of ectopically expressed genes on thymocyte development was assessed flow cyto- despite the absence of DP thymocytes, the gd T cells that devel- metrically by gating on cells positive for the fluorescent indicator protein. oped in these models were functional and expressed several of DN3 cells (d7 HSC OP9-DL1 cultured) were transduced by overnight coculture the genes comprising the ‘‘gd-biased gene profile’’ (Pennington using stable retrovirus-producing GP+E.86 packaging cells as previously et al., 2003; Silva-Santos et al., 2005). Interestingly, we found described, followed by coculture on OP9 monolayers in OP9-medium

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(a-MEM supplemented with 1 ng/ml mouse IL-7 [Peprotech] and 5 ng/ml Animal. We acknowledge the Sunnybrook Research Institute Centre for human recombinant Flt-3 ligand [Peprotech]) (Ciofani et al., 2006). Cytometry and Scanning Microscopy and the Division of Comparative Research. This work was supported by NIH grants (D.L.W.) AI081314, NIH Flow Cytometry core grant P01CA06927, Center grant no. P30-DK-50306, and an appropria- Skin preparations were performed as described (Havran et al., 1991). All tion from the Commonwealth of Pennsylvania; and by a grant (J.C.Z.-P.) single-cell suspensions were stained with commercially available Ab (BD Phar- from the Canadian Institute of Health Research (CIHR-MOP42387). J.C.Z.-P. Mingen, eBiosciences, and Caltag) and analyzed on either a FACSVantageSE is supported by a Canada Research Chair in Developmental Immunology. (Becton Dickinson) or BDLSR using Flowjo software (Treestar, Inc.). Anti-Vg Ab G.W.W. and M.C. were supported by a CIHR Scholarship. J.P.L. and S.Y.L. were generously provided by Dr. P. Pareira (Pasteur Institute). BrdU staining were supported by the Fox Chase Cancer Plain and Fancy and Greenwald was performed according to manufacturer’s specifications following a 4 hr Postdoctoral Fellowships, respectively. pulse with BrdU (i.p. injection of 100 mg/mouse). Where applicable, dead cells were excluded from the analyses using propidium iodide (PI) or DAPI gating. Received: December 15, 2008 Revised: May 27, 2009 Real-Time PCR Accepted: July 28, 2009 Thymocyte populations were purified by flow cytometry, where necessary, Published: October 15, 2009 following depletion using magnetic anti-CD8 beads (Miltenyi Biotec). RNA was extracted using the RNeasy kit according to manufacturer’s specifications (QIAGEN) and converted to cDNA using the SuperscriptII kit (Invitrogen). 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