Inhibitor of DNA Binding 3 Limits Development of Murine Slam-Associated Adaptor Protein-Dependent “Innate” γδ T cells

Background Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the αβ and γδ T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRαβ+ T lymphocytes. More recently, Id3 −/− mice on a C57BL/6 background were shown to have a dramatic expansion of γδ T cells. Methodology/Principal Findings Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of γδ T cells that express a Vγ1.1+Vδ6.3+ receptor with restricted junctional diversity. These Vγ1.1+Vδ6.3+ T cells have multiple characteristics associated with “innate” lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other “innate” lymphocyte populations, development of Id3 −/− Vγ1.1+Vδ6.3+ T cells requires the signaling adapter protein SAP. Conclusions Our data provide novel insight into the requirements for development of Vγ1.1+Vδ6.3+ T cells and indicate a role for Id3 in repressing the response of “innate” γδ T cells to SAP-mediated expansion or survival.


Introduction
T lymphocytes bearing ab or cd T cell receptors (TCR) develop in the thymus from a common progenitor cell pool. Most cells in the adult thymus express the co-receptor molecules CD4 and CD8 and represent an intermediate stage in ab T cell development that has undergone productive TCRb rearrangement and is in the process of TCRa rearrangement. After expression of a functional TCRa CD4 + CD8 + (double positive, DP) cells undergo negative or positive selection and become single positive (SP) cells [1]. In contrast, the earliest T cell progenitors, and cd T cells, are CD4 2 CD8 2 (double negative, DN) and can be divided into four stages based on expression of CD117 (c-kit) and CD25; DN1, (c-kit + CD25 2 ), DN2 (c-kit + CD25 + ), DN3 (c-kit-CD25 + ) and DN4 (c-kit-CD252) [2]. DN1 and DN2 cells are the most immature T cell progenitors and are not yet fully committed to T cell differentiation [3,4]. Rearrangement of TCR loci initiates at the DN2 stage but is most prevalent in DN3 cells [5,6]. DN3 cells that rearrange and express TCRb undergo b-chain selection and progress to the DN4 stage before becoming DP [7]. In contrast, cells that rearrange functional Tcrg and Tcrd genes diverge from the ab pathway and become DN cd T cells [2]. The stage at which the ab and cd T cell lineages diverge remains controversial [8,9,10,11,12].
During ontogeny, the variable gene segments of the Tcrg and Tcrd genes are rearranged in ordered waves. The first wave occurs around embryonic day 13 and includes rearrangement of Vc3 and Vd1 and is followed by rearrangement of Vc4 [13,14] (Nomenclature according to [15]). These receptors contain limited diversity at the junction of the V, diversity (D), and joining (J) segments [16,17] in part because terminal deoxylnucleotidyl transferase (TdT), a polymerase that adds non-templated nucleotides, is absent from embryonic cells [18,19]. Consequently, the first cd T cells express invariant Vc3/Vd1 or Vc4/Vd1 TCRs and home specifically to the epidermis or the epithelium of the reproductive tract and the tongue, respectively [20]. In contrast to the embryo, the adult thymus rearranges Vc1.1, Vc2 and Vc5 and generates receptors with extensive junctional diversity, creating a highly diverse cd TCR repertoire [21]. Interestingly, a subset of cd T cells with an invariant Vc1.1 + Vd6.3 + TCR has been described that resides in the adult thymus, spleen, and liver [22]. These cd T cells develop from late embryonic precursors and expand during neonatal life [23]. Vc1.1 + Vd6.3 + T cells share multiple characteristics with natural killer (NK) T cells including expression of the activation markers CD44, and NK1.1, and low expression of the immature T lymphocyte marker CD24. Moreover, both NKT and Vc1.1 + Vd6.3 + T cells secrete IFNc and IL4 rapidly after stimulation in vitro [24]. These findings led to the hypothesis that NKT and Vc1.1 + Vd6.3 + T cells represent innate branches of the ab and cd T cell lineages, respectively [25]. The presence of an invariant receptor on these two T cell subsets is consistent with the hypothesis that the functional characteristics of these ''innate-like'' cells are determined in part via selection by endogenous ligands.
T cell development is intimately linked to activity of the E protein transcription factors E2A and HEB [26,27,28]. E proteins are essential at multiple stages of ab T cell development and function in lymphocyte survival, proliferation and differentiation. Importantly, induction of E protein antagonists such as Id2 and Id3 appears to be critical for b-selection and positive selection of ab T cells [29,30,31]. Cross-linking of CD3e (a component of the TCR signalling complex) on DN3 thymocytes induces Id3 through a MAP kinase-dependent pathway [32]. Mice lacking Id3 show mild defects in positive selection similar to those observed in mice lacking the Tec kinase Itk, which activates the MAP kinases Erk1 and Erk2 [33,34]. MAP kinase signalling is also important for proper cd T cell development and Id3 is highly expressed in cd T cells, although published data suggest that Id3 is not essential for cd T cell development in mice expressing the KN6 (Vc2 + Vd5 + ) transgene [10]. Surprisingly however, it was reported that Id3 2/2 mice have an increased number of cd T cells and it was hypothesized that Id3 functions in DN3 cells to prevent Tcrg or Tcrd rearrangement in cells expressing a functional TCRb [35].
Here we report that the elevated number of cd T cells in Id3 2/2 mice is a consequence of an expanded population of Vc1.1 + Vd6.3 + T cells. Id3 2/2 Vc1.1 + Vd6.3 + T cells, like their wild-type (WT) counterparts, primarily develop from late embryonic or neonatal progenitors rather than adult DN3 cells. These cd T cells have many of the characteristics of NKT cells previously noted, and we confirm that Id3 2/2 Vc1.1 + Vd6.3 + T cells express the transcription factor promyelocytic leukemia zinc finger (PLZF) protein [36], a molecular determinant of the NKT cell fate [37,38] and their development required the Signaling lymphocyte adaptor molecule (Slam)-associated Adaptor Protein (SAP) [39]. Importantly, deletion of SAP overcomes all apparent thymic alterations in Id3 2/2 mice including the increased number of cd T cells and the reduced thymic cellularity, whereas deletion of Tcrd had no effect on thymic cellularity. These observations indicate that Id3 plays a role in preventing expansion or survival of this SAP-dependent lymphocyte. Taken together, our data demonstrate that Id3 functions to limit the development of SAP-dependent ''innate-like'' cd T cells.

Results
Development of CD4 + and CD8 + cd T Cells in Id3 2/2 Mice While investigating the thymic phenotype of Id3 2/2 mice we discovered that the number of cd T cells is increased by approximately 8-fold (range 3-to 15-fold) compared to Id3 +/+ mice ( Fig. 1A and B). In contrast, the frequency of TCRb high cells was similar among Id3 +/+ and Id3 2/2 thymocytes, although the number of TCRb + cells is decreased in the absence of Id3 since Id3 2/2 mice have a 3-fold decrease in thymocytes numbers ( Fig. 1A and Fig. S1). Further analysis revealed that a large portion of Id3 2/2 TCRc + cells express CD4 or CD8 (Fig. 1C). Compared to Id3 +/+ mice, Id3 2/2 mice have an increased number of CD4 (80-fold) and CD8 (70-fold) TCRc + cells as well as DN (5.5-fold) and DP (5-fold) TCRc + cells (Fig. 1D). Id3 2/2 TCRc + cells express CD8 as a CD8aa homodimer as opposed to the CD8ab heterodimer expressed by TCRb + CD8 + cells (Fig. 1E). Importantly, Id3 2/2 TCRc + cells expressed significantly more mRNA for the transcription factor Sox13 than DP thymocytes indicating that these are bona fide cd T cells [40] (Fig. 1F). In the spleen there is also a large population of TCRc + cells expressing CD4 or CD8aa that is markedly elevated compared to Id3 +/+ mice (Fig. S2). Taken together these data indicate that Id3 limits development of cd T cells, in particular, cd T cells expressing CD4 or CD8.
Id3 2/2 cd T Cells Have an Activated or ''Innate-Like'' Phenotype In light of our observations that Id3 2/2 cd T cells expressed CD4 and CD8, we characterized these cells for expression of multiple cell surface proteins. In the thymus, the majority of Id3 2/2 DN TCRc + cells had high expression of CD122, NK1.1 and CD44 and low expression of CD24 compared with Id3 +/+ TCRc + cells ( Fig. 2A and B). This phenotype is associated with activation of ab and cd T cells [41,42,43]. Notably, a majority of the Id3 2/2 NK1.1 + TCRc + cells expressed TCRc at low levels ( Fig. 2B). A subset of cd T cells expressing NK1.1 with low expression of TCRcd is present in the spleen of WT mice and presumably represent activated cd T cells [42]. Similar to Id3 2/2 DN TCRcd + cells, a portion of Id3 2/2 CD4 and CD8 cd T cells had these activation markers, although the CD4 cells had lower levels of CD122 and only a small portion expressed NK1.1 (Fig. 2B). Therefore, our data indicate that a large portion of the cd T cells in the thymus of Id3 2/2 mice have an activated phenotype. The DN, CD4 and CD8 cd T cells in the spleen of Id3 2/2 mice also have an activated phenotype (Fig. S3).
A subset of CD122 + cd T cells, which are thought to have encountered ligand in the thymus, produce IFNc rapidly after in vitro stimulation [43]. To determine whether Id3 2/2 cd T cells represent previously activated cells we tested their ability to make IFNc after in vitro stimulation with PMA and ionomycin for 5 hours. Importantly .30% of Id3 2/2 TCRc + thymocytes produce IFNc under these conditions. In contrast only 2.5% of Id3 +/+ TCRc + thymocytes produced IFNc at this early time point (Fig 2C). Similarly, more than 50% of Id3 2/2 splenic TCRc + cells produced IFNc ( Fig S3). Interestingly, a subset of Id3 2/2 cd T cells make both IFNc and IL-4 ( Fig. S4). Cytometric bead analysis revealed that Id3 2/2 cd T cells also make more IFNc, IL4, IL10 and IL13 than their WT counterparts after stimulation with anti-TCRc (Fig. S4). Taken together, our data demonstrate that Id3 2/2 mice develop a large population of cd T cells that show characteristics of previously activated cells.

Id3 2/2 cd T Cells with an Activated Phenotype Develop Early in Post-Natal Life
To determine when during ontogeny Id3 2/2 cd T cell numbers increase and when the activated phenotype becomes evident, we examined thymocytes from mice isolated 1 week after birth. At this stage of ontogeny, few thymocytes have left the thymus and therefore peripheral activation is unlikely to have impacted on thymocyte numbers or phenotype. Importantly, a 10-fold increase in TCRc + cells was observed in Id3 2/2 neonates and the aberrant expression of CD4 was already evident (Fig. 3A, B and C). Moreover, Id3 2/2 neonatal cd T cells had an activated phenotype similar to that observed in the adult Id3 2/2 thymus ( Fig. 3D and E), although only a small subset of these cells were positive for NK1.1. Taken together, our data indicate that Id3 limits the development of cd T cells with an activated phenotype in neonatal mice. Our data also indicate that the activated phenotype of Id3 2/2 cd T cells likely occurs within the thymus rather than as a consequence of peripheral activation since few thymocytes have left the thymus within the first week after birth [44].
The Majority of Id3 2/2 cd T Cells Express Vc1.1 and Vd6.3 The presence of a large population of activated cd T cells in the Id3 2/2 neonatal thymus suggests that these cells derive from cells that underwent V(D)J recombination in the late embryonic or neonatal period. To gain insight into the origin of the majority of Id3 2/2 cd T cells, we examined their TCR repertoire by staining with a panel of anti-Vc antibodies. This analysis revealed that .90% of cd T cells in the Id3 2/2 thymus express Vc1.1 (Fig. 4A,   B). This increase in Vc1.1 usage is not at the expense of the other Vc gene segments since the total number of Vc2 + and Vc5 + cd T cells are similar to that in the WT thymus, although their frequency within the cd T cell population is reduced (Fig. 4C).
Importantly, the majority of Vc1.1 + cells in the Id3 2/2 thymus co-express Vd6.3 (Fig. 4A). Vc1.1 + Vd6.3 + cd T cells have been reported to be of late fetal origin, express CD4 and have an activated phenotype including high expression of CD44, low expression of CD24 with rapid production of IFNc and IL4 similar to what we have observed with Id3 2/2 cd T cells [22]. These observations lead us to conclude that Id3 deficiency allows for an increase in the number of Vc1.1 + Vd6.3 + cd T cells without a major effect on Vc2 + or Vc5 + cd T cells. Figure 1. Altered cd T cell development in Id3 2/2 mice. A) FACS analysis for TCRb and TCRcd on Id3 +/+ and Id3 2/2 thymocytes. The frequency of TCRb + cells and TCRcd + cells is shown. B) Number of TCRcd + cells in the thymus of Id3 +/+ and Id3 2/2 mice. Average +/2 standard deviation was determined from .15 mice. p,0.0005. C) CD4 and CD8 expression on TCRcd + cells. D) Number of CD4, CD8, DN and DP TCRcd + cells in the thymus Id3 +/+ (grey) and Id3 2/2 (black) mice. Average +/2 standard deviation was determined from .15 mice. p,0.0005 in all Id3 +/+ to Id3 2/2 comparisons. E) Analysis of Id3 2/2 CD8a + thymocytes for TCRb and CD8b (left panel) or TCRc (right panel) and CD8b. The TCRb + cells express CD8b whereas TCRcd + cells are CD8b 2 (right panel) and presumably CD8aa. F) QPCR for Sox13a mRNA in sorted Id3 +/+ (grey) and Id3 2/2 (black) TCRc + cells and DP thymocytes (standardized to Hprt). Bars are the average from 3 experiments +/2 standard deviation. doi:10.1371/journal.pone.0009303.g001 Limited Diversity in Vc1.1-Jc4 and Vd6-Jd1 Rearrangements in Id3 2/2 cd T Cells In WT mice Vc1.1 + Vd6.3 + T cells develop from fetal precursors that rearrange the c and the d chains in late embryonic life [23]. These cells show frequent rearrangement of the Vc1.1 variable gene segment to the Jc4 joining segment and of Vd6.3 to Jd1 and, depending on the genetic background of the mice, can have oligoclonal or polyclonal junctional sequences [45,46]. To gain insight into the complexity of the rearrangements in Id3 2/2 Vc1.1 + Vd6.3 + cells we amplified and sequenced the Vc1.1-Jc4 and Vd6-Jd1 junctions in the TCRc + population. Analysis of Vc1.1-Jc4 junctions revealed that 30 of 31 sequences were inframe and consisted of only two unique sequences indicating a population of Vc1.1 + T cells lacking significant TCR diversity. In addition, these sequences lacked N nucleotide additions suggesting that the rearrangements occurred in the absence of TdT (Fig. 5A). Analysis of Vd6-Jd1 junctions also revealed a lack of diversity with 32 of 37 in-frame sequences containing the Vd6.3 gene segment, consistent with our flow cytometry analysis (Fig. 5B). Moreover, 21 of the 32 Vd6.3-Jd1 junctions are represented by only two sequences. In the majority of sequences the Dd2-Jd1 and Vd6-Dd2 junctions resulted in maintenance of the germline sequence and the Dd1 gene segment was not observed in these junctions (Fig. 5B). Of the 4 unique sequences that showed diversity following the Vd6.3 gene segment at least 2 represent potential P rather than N nucleotide additions. Notably, the invariable Dd2-Jd1 junction forces a unique reading frame of the Dd2 segment (V/IGGIRA), which contributes to the CDR3 domain [47], thus resulting in a highly invariant Vc1.1 + Vd6.3 + TCR, at least for those cells using the Vc1.1-Jc4 and Vd6-Jd1 rearrangement. The presence of a highly invariant receptor on cells with an activated phenotype suggests that the Vc1.1 + Vd6.3 + T cells are selected by a ligand present in the thymus.
To examine the possibility that the Vc1.1 + Vd6.3 + T cells in Id3 2/2 mice arise as a consequence of preferential Vc1.1 and Vd6.3 rearrangement in adult thymocytes we analyzed the Vc1.1-Jc4 and Vd6-Jd1 junctions in unselected Id3 2/2 DN3 cells. This analysis revealed that the in-frame Vc1.1-Jc4 rearrangements (8/15) contained 3 unique sequences that were distinct from those amplified from Id3 2/2 cd T cells (Fig. 5A). In addition, only 3 of 16 Vd6-Jd1 junctions were in-frame and each of these sequences was unique with one sequence containing the Vd6B gene segment (Fig. 5B). Therefore, Id3 2/2 DN3 cells show no evidence of a preferential production of the Vc1.1-Jc4 or Vd6.3-Jd1 junctions used in the cd T cells in Id3 2/2 mice. Further, if a small number of Vc1.1 + Vd6.3 + cd T cells with this rearrangement developed in the adult and expanded we would expect this population of cd T cells to incorporate more BrdU than WT cd T cells. However, multiple in vivo BrdU incorporation experiments failed to reveal an increase in proliferation of Id3 2/2 cd T cells (Fig. S5) [35]. Taken together, our results indicate that in the absence of Id3 there is an elevated number of Vc1.1 + Vd6.3 + cd T cells that originate during late fetal or neonatal life. Consistent with this conclusion, reconstitution of WT or Id3 2/2 mice with adult Id3 2/2 bone marrow hematopoietic stem and progenitor cells largely fails to reconstitute this cd T cell population (Fig. S6).

Development of Activated cd T Cells in Id3 2/2 Mice Requires SAP
A subset of Vc1.1 + Vd6.3 + , referred to as cd NKT, share phenotypic and functional characteristics with NKT cells including expression of the transcription factor PLZF and a requirement for SAP-dependent [24,37,39,48]. However, some Vc1.1 + Vd6.3 + T cells develop independent of SAP signaling [24]. To further establish the parallels between Id3 2/2 Vc1.1 + Vd6.3 + cells and NKT cells we investigated the expression of PLZF. Importantly, PLZF was highly expressed in these cells compare to Vc1.1 2 Vd6.3 2 cd T cells, in both the Id3 +/+ and the Id3 2/2 thymus (Fig. 6A). We examined whether development of this activated cd T cell population in Id3 2/2 mice requires SAP by generating Id3 2/2 Sh2d1a 2/2 mice. Strikingly, the total number of cd T cells in Id3 2/2 Sh2d1a 2/2 mice was similar to that in WT and Sh2d1a 2/2 mice ( Fig. 6B and C). Moreover, the frequency of Vc1.1 + Vd6.3 + cd T cells in Id3 2/2 Sh2d1a 2/2 mice was similar to WT and Id3 2/2 Sh2d1a 2/2 cd T cells showed no evidence of an activated phenotype (Fig. 6D and Fig. S7). Therefore, activation of the SAP signaling pathway is essential for the cd T cell phenotype observed in Id3 2/2 mice. These data indicate that SAP is essential for development or survival of the Vc1.1 + Vd6.3 + T cells present in Id3 2/2 mice. Interestingly, all of the observed alterations in the Id3 2/2 thymus were normalized by deletion of Sh2d1a. That is, cd T cell numbers and phenotype as well as total thymocytes numbers are similar to WT in Id3 2/2 Sh2d1a 2/2 mice (Fig. S7). This finding is striking because deletion of cd T cells in Id3 2/2 mice, by creating Id3 2/2 Tcrd 2/2 mice, does not restore thymic cellularity to WT levels (Fig. S8). Therefore, multiple alterations in the Id3 2/2 thymus are dependent on SAP signaling. Id3 is a transcriptional repressor that prevents E proteins from binding DNA [49]. All of the E proteins are expressed in T cells; however, deletion of E2A is sufficient to restore ab T cell maturation defects in Id3 2/2 [50]. Therefore, we tested the requirement for E2A in the development of activated cd T cells in Id3 2/2 mice by generating Id3 2/2 E2A 2/2 mice. Consistent a previous study we found that E2A is required for development of normal numbers of cd T cells (Fig. 7A and B) [51]. Importantly, mice that lack both Id3 and E2A have fewer cd T cells than WT mice but more cd T cells than E2A 2/2 mice (Fig. 7A and B). Nonetheless, the cd T cells that develop in Id3 2/2 E2A 2/2 mice fail to express CD122 and NK1.1 (Fig. 7C) Therefore, E2A is required for the development of cd T cells with an activated phenotype in Id3 2/2 mice.

Discussion
In this study, we report that Id3-deficiency results in a 8-fold increase in the number of cd T cells in the thymus and that the majority of these cells likely express an invariant Vc1.1 + Vd6.3 + TCR. Similar to WT Vc1.1 + Vd6.3 + cells, Id3 2/2 cd T cells have high expression of CD122, CD44 and NK1.1, low expression of CD24, and rapidly secrete IFNc and IL4 after in vitro stimulation. The ''activated'' phenotype of these cd T cells parallels that of NKT cells, a finding that has led to the hypothesis that on the right) or DN3 thymocytes (lower, indicated on the right). Data are cumulative from 2 independent experiments in which DNA was isolated 50,000 TCRcd + cells and 30,000 Lin 2 c-kit 2 CD25 + (DN3) cells. In each experiment 3 independent PCR amplifications were performed on each population and cloned into pBSK for sequencing. A minimum of 30 sequences were analyzed for each population. Results from the two experiments were essentially identical. Only in-frame sequences are shown and the number of clones sharing the identical sequence is indicated on the right in parenthesis. The amino acid sequence is shown in bold below the DNA sequence. The Vd6.3 primer also amplifies the Vd6B gene segment and sequences derived from Vd6B are indicated on the right. P/N represent potential P or N additions and the underlined sequences are potential P additions. Sequences derived from the Dd1 and Dd2 gene segments are also indicated. doi:10.1371/journal.pone.0009303.g005 Vc1.1 + Vd6.3 + T cells represent an innate branch within the cd T cell lineage [25]. Here, we demonstrate that both Id3 +/+ and Id3 2/2 Vc1.1 + Vd6.3 + cells express the transcription factor PLZF, a molecular determinant of the NKT cell fate [37]. Moreover, we find that SAP is essential for development of Id3 2/2 Vc1.1 + Vd6.3 + T cells, as is the case for NKT cells [39,52,53]. We, and others, have found that the majority of adult cd T cells in Id3 2/2 mice proliferate at a rate similar to WT cd T cells indicating that the Id3 2/2 Vc1.1 + Vd6.3 + population is not increased because of extensive proliferation in the adult thymus [35], rather, we conclude that these cd T cells expand during neonatal life. Our data are consistent with a model in which Id3 controls the response of Vc1.1 + Vd6.3 + T cells to ligand-and/or SAP-mediated proliferation.
We demonstrate that the increased number of cd T cells in Id3 2/2 mice is attributed to the increase in SAP-dependent cells. Deletion of Sh2d1a in Id3 2/2 mice abrogated the increase in cd T cell numbers and the activated phenotype. Therefore, the major effect of Id3-deficiency on cd T cell development is an increase in embryonically derived Vc1.1 + Vd6.3 + T cells. This conclusion is in contrast to a previous report suggesting that alterations in adult DN3 cells underlie the increased production of cd T cells in Id3 2/2 mice [35]. This conclusion was based, in part, on the observation that Id3 2/2 cd T cells have less germline DNA at the TCRb locus than WT cd T cells. This finding led the authors to conclude that the cd T cells developing in Id3 2/2 mice derive from cells that have an extended opportunity for TCRb rearrangement. Our findings suggest that the reason for the increased TCRb rearrangement may stem from differences in fetal versus adult cells rather than differences in Id3 +/+ and Id3 2/2 adult DN3 cells. Our findings are also inconsistent with a model in which Id3 plays a critical role in selection of self-ligand reactive cd T cells; however, many of the Id3 2/2 Vc1.1 + Vd6.3 + cells express CD4 or CD8 which is consistent with a failure to prevent some aspects of ab T cell development [54]. More importantly, our data reveal that SAP-dependent signaling pathways are critically linked to the altered phenotype of Id3 2/2 T cells since the thymus of Id3 2/2 Sh2d1a 2/2 mice, unlike the Id3 2/2 or the Id3 2/2 Tcrd 2/2 thymus, is indistinguishable from the Id3 +/+ or Shld1a 2/2 thymus with respect to cellularity and phenotype.
Our hypothesis that Id3 functions downstream of TCR signals to limit SAP-dependent proliferation in cd T cells is consistent with previous studies demonstrating that Id3 is a target of TCR triggered signaling in both ab and cd T cells [30,32]. The pathway from the TCR leading to Id3 involves the MAP kinases Erk1 or Erk2, which are triggered by the Tec kinases Itk and Rlk [55]. Recently, Itk 2/2 mice were reported to have an increased number of PLZF-expressing Vc1.1 + Vd6.3 + T cells, implying that Itk may also limit development of ''innate'' cd T cells [25,56]. Our data are consistent with the hypothesis that Id3 is an essential effector of the TCR-Itk-MAP kinase pathway that determines the consequence of signaling through the Vc1.1 + Vd6.3 + TCR.
Id3 is an inhibitor of E protein DNA binding [57]. We, and others, found that deletion of E2A in Id3 2/2 mice blunted the development of activated cd T cells indicating that elevated E2A function is critical for development of these cells [35]. It should be noted that E2A is required for normal cd T cells development and affects the timing of rearrangement of specific Vc receptors [21,51]. Therefore, loss of activated cd T cells in E2A 2/2 Id3 2/2 mice, as compared to Id3 2/2 mice, could be the result of E2A functions upstream of TCR signaling and independent of Id3. However, it seems likely that Id3 deletion leads to heightened E2A (or E protein) activity after TCR-initiated signaling events, where E2A activity would normally be inhibited. In the case of cd T cells, elevated activity of E2A may cooperate with SAP-dependent signals to promote an outcome from TCR-mediated signaling that is not typical, for example, leading to prolonged survival or proliferation.
Our analysis of Vc1.1-Jc4 and Vd6-Jd1 sequences in Id3 2/2 cd T cells and DN3 thymocytes lead us to conclude that the majority of Vc1.1 + Vd6.3 + cells in Id3 2/2 mice develop during fetal or neonatal life. This analysis revealed that Id3 2/2 cd T cells have germline sequences at the Vc1.1-Jc4 and Dd2-Jd1 junctions, very low diversity in the Vd6.3-Dd2 junction and complete absence of the Dd1 segment. However, Vc1.1-Jc4 and Vd6-Jd1 sequences retrieved from Id3 2/2 DN3 progenitors are characterized by diverse junctions. It is possible that the Vc1.1-Jc4 and Vd6-Jd1 sequences observed in Id3 2/2 cd T cells could be generated from adult DN3 cells and that thymic selection leads to expansion of these cells. However, two observations argue against this possibility. First, Id3 2/2 cd T cells proliferate to a similar extent as WT cd T cells in the adult thymus and second, development of Vc1.1 + Vd6.3 + T cells is blunted in WT or Id3 2/2 mice reconstituted with Id3 2/2 adult bone marrow. Therefore, adult thymic progenitors do not efficiently recapitulate the cd T cell phenotype observed in Id3 2/2 mice. The activated phenotype of Id3 2/2 Vc1.1 + Vd6.3 + T cells is consistent with the hypothesis that this receptor recognizes a ligand in the thymus. cd T cells that recognize the unconventional MHC molecule T10-or T22 and Vc3 + Vd1 + DETCs, which are also hypothesized to be ligandselected have a similar phenotype [58].
Our results reveal an important role for Id3 in limiting the number of Vc1.1 + Vd6.3 + T cells. Since Vc1.1 + Vd6.3 + T cells share many features with NKT cells including rapid production of IFNc and IL-4, their increased numbers could significantly alter immune responses. Indeed, Itk 2/2 mice, which also have an increased number of Vc1.1 + Vd6.3 + T cells, have elevated serum IgE that is dependent on cd T cells [25,56]. Therefore, while Id3 appears to be largely dispensable for development of conventional cd T cells, it limits the number of PLZF-expressing SAPdependent ''innate'' cd T cells.

Ethics Statement
All animal experiments were performed in compliance with the requirements of the University of Chicago Institutional Animal Care and Use Committee

Mice
Mice were housed at The University of Chicago Animal Resource Center. Id3 2/2 and Tcrd 2/2 mice were purchased from Jackson ImmunoResearch. Sh2d1a 2/2 mice were a kind gift from C. Terhorst. Genotyping was as previously described [59,60,61]. All experiments were performed on mice that were 6 to 8 weeks old unless otherwise indicated.