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 γδ-lineage cells independent of Notch signals and functionally competent. Id3 is required for strong signals to promote adoption of the γδ-lineage and oppose adoption of the αβ-fate. We recently reported that γδ-lineage cells are far less dependent on Notch signaling than αβ-lineage cells (Ciofani et al., 2006
). Based on our present findings, it is now clear that Id3 is both necessary and sufficient for Notch independence of γδ-lineage cells; and that this independence requires a functional helix through which Id3 heterodimerizes with, and suppresses the function of, E proteins. Finally, Id3 is also necessary and sufficient to arm γδ-lineage cells for effector function defined by TCR- or mitogen-induced proliferation and IFNγ production. These findings place Id3 as a central molecular mediator of the strong signals that influence T cell fate, as well as their developmental and functional characteristics.
It has been suggested for some time that αβ- and γδ-lineage precursors are differentially dependent upon Notch signaling, though this has been controversial until recently (Garcia-Peydro et al., 2003
; Hernandez-Hoyos and Alberola-Ila, 2003
; Radtke et al., 2004
; Washburn et al., 1997
). Indeed, we showed that while αβ-lineage precursors remain Notch-dependent during their pre-TCR induced transition to the DP stage, γδ-precursors become independent of Notch signaling upon expression of the γδTCR (Ciofani et al., 2006
). Nevertheless, it was unclear how γδTCR signaling permitted Notch-independent differentiation of γδTCR-expressing DN cells. We now show that Id3 is an important molecular effector of this process. The molecular basis for this effect has not been established; however, recent evidence from the Murre lab (as well as genetic analysis from Drosophila
) suggests interplay between E proteins and the Notch pathway (Ikawa et al., 2006
; Schwartz et al., 2006
). Moreover, numerous reports support the notion that suppression of E protein function (at least transiently) is required for early thymocyte differentiation (Engel and Murre, 2004
; Koltsova et al., 2007
; Murre, 2005
). We now propose a model whereby lineage commitment and Notch-dependence are determined by graded reductions in E protein activity mediated by differences in TCR signal strength (Figure S9
). DN3 thymocytes are prevented from further development by E protein-mediated enforcement of the β-selection checkpoint, as evidenced by the ability of E2A-deficiency to enable pre-TCR deficient thymocytes to traverse the β-selection checkpoint and differentiate to the DP stage (Engel and Murre, 2004
; Wojciechowski et al., 2007
). Paradoxically, E protein deficiency blocks the development of pre-TCR expressing cells beyond the β-selection checkpoint to the DP stage, suggesting that the induction of αβ-lineage development by pre-TCR signals is dependent upon partial or temporally-restricted suppression of E protein activity (Engel 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 αβ-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 degradation (Nie et al., 2003
; Ordentlich et al., 1998
) and through induction of Id3, which can directly repress E protein function (Reynaud-Deonauth et al., 2002
). Unlike the weak signals that promote αβ-lineage development, the strong signals that confer Notch-independent differentiation upon γδ-lineage cells are dependent upon Id3 induction and are sufficient to suppress E protein activity beyond the threshold required for γδ-lineage commitment and development without assistance from Notch activity.
Because effects on growth and survival are associated with the regulation of lineage fate by Id3 in the context of strong TCR signals, it is possible that Id3 is not affecting lineage fate per se, but rather is affecting the growth and survival of pre-committed αβ or γδ lineage progenitors. Nevertheless, a number of lines of evidence argue against this perspective and for a role of Id3 in influencing lineage commitment. If strong signals were simply eliminating αβ-lineage cells, then Id3 transduction would be predicted to preferentially eliminate pre-TCR expressing αβ-lineage progenitors; however, Id3-transduction does not do so (Figure S4
). Regarding the promotion of the γδ fate by strong signals (or Egr-transduction), the converse argument could be made that strong signals promote the generation of CD24lo
γδ-lineage cells by preferentially expanding a small pre-committed population. However, we demonstrated that ectopic expression of the KN6 γδTCR in Rag2-/- thymocytes (which lack pre-committed γδ-lineage cells) promoted the γδ fate and opposed the αβ fate in the presence of ligand and produced the converse effect in the absence of ligand (Figure S5
). It should be noted that this represents the first example where the expression of a specific γδTCR ligand was demonstrated to be required for selection and development of γδ-lineage precursors. Altogether, these data suggest that strong signals and Id3 drive lineage commitment.
While we had previously reported impaired development of γδ T cells in fetal Id3-deficient mice, the role of Id3 as a critical molecular effector in γδ cell specification and development is also revealed by the perturbation of γδ development in adult Id3-deficient mice. A recent report suggested that Id3 functions to restrain the development of γδ-lineage cells, as γδ T cell numbers were increased in adult Id3
-deficient mice (Ueda-Hayakawa et al., 2009
). The expansion was accompanied by enhanced V(D)J recombination at the TCR loci (γ,δ, and β) and an outgrowth of the Vγ1.1+
γδ cell subset. We observed similar effects of Id3-deficiency, but propose an alternative interpretation. We suggest that Id3-serves to terminate V(D)J recombination following TCR expression (either pre-TCR or γδTCR expression), thereby limiting the developmental time window during which TCR rearrangement can occur and explaining why TCR rearrangements were more extensive in Id3
-deficient mice. We further demonstrated that while γδ T cell numbers were increased in Id3-defcient mice, the expansion appeared to be restricted to the Vγ1.1+
subset, as the Vγ2 and Vγ3 γδ subsets were severely reduced. We think these differential effects on particular Vγ subsets relate to their respective affinities for selecting ligands, with Id3-deficiency impairing selection of those with intermediate affinity, while allowing those of high affinity to escape deletion. This interpretation is supported by our demonstration that Id3-deficiency impairs selection of KN6 γδTCR Tg thymocytes on the intermediate affinity T-22d
ligand, while rescuing them from deletion by T-10/22b
, for which their affinity is 10-fold higher (Adams et al., 2008
). Indeed, at least some Vγ1.1+
γδ cells, which are expanded in Id3
-/- mice, are thought to belong to an NK γδ T cell lineage selected on high affinity ligand (Felices et al., 2009
; Lees et al., 2001
; Qi et al., 2009
). Taken together, these findings suggest an apparent dichotomy of Id3 function, restricting production of γδ cells whose TCR affinity for ligand is very high, while promoting differentiation of the broader repertoire of non-autoreactive γδ-T cells.
Along with its role in promoting the Notch independence of developing γδ T cells, Id3 plays an important role in the acquisition of effector function by γδ-lineage cells. We found that Id3
-deficiency impairs the ability of KN6 γδTCR Tg+
cells to proliferate and produce IFNγ in response to TCR ligation. Conversely, ectopic expression of Id3 in the absence of TCR expression, conferred the ability to proliferate and produce IFNγ in response to mitogenic signals. The Hayday lab has reported that developing γδ T cells require presentation of LTβ in trans
by DP thymocytes for both functional competence and the expression of a number of genes termed the “γδ-biased gene profile” (Pennington et al., 2003
; Silva-Santos et al., 2005
). DP thymocytes are not present in appreciable numbers in either the ligand-expressing KN6 γδTCR Tg mice or among Id3
-transduced DN3 cells cultured on OP9-monolayers in vitro. Nevertheless, despite the absence of DP thymocytes, the γδ T cells that developed in these models were functional and expressed several of the genes comprising the “γδ-biased gene profile” (Pennington et al., 2003
; Silva-Santos et al., 2005
). Interestingly, we found that Id3
-deficiency separated γδ-function from expression of the γδ-biased gene profile in that the Id3
-deficient γδ cells expressed a subset of the profile genes, with the notable exception of Rgs1, and yet were not functional (Figures and ). One possible explanation is that only a subset of the profile genes is uniquely associated with γδ-function. For example, although the function of Rgs1 in T cells is unknown, our data suggest that Rgs1 expression is tightly correlated with γδ T cell function, raising the interesting possibility that Rgs1 plays an active role in promoting the functional competence of γδ T cells.
The role of Id3 in promoting functional competence also provides an important mechanistic insight into a recent report regarding the role of ligand in shaping γδ T cell effector function. The report contends that ligand-mediated selection in the thymus does not play a significant role in shaping the γδ TCR repertoire but does influence the nature of effector function (Jensen et al., 2008
). That is, γδ T cells that purportedly did not encounter a selecting ligand during development (antigen-naïve) are programmed to produce IL-17, whereas antigen-experienced γδ T cells produce IFNγ. While ruling out the possibility of ligand-mediated selection in the thymus is extremely difficult, we would agree that ligand-mediated selection arms γδ T cells to produce IFNγ (Figures and ). Indeed, our data supports the notion that the induction of Id3 following TCR-ligand engagement plays a critical role.
Taken together, our findings place Id3 as a central molecular mediator of the strong signals that influence T cell fate, enable developing γδ T cells to gain Notch-independent maturation, and shape their functional attributes. While it is likely that Id3 (perhaps with assistance from Id2) mediates these effects by suppressing the function of E proteins, this interpretation remains to be fully characterized. Future efforts will be directed at determining how the ERK-Egr-Id3 pathway, as well as other mediators of strong signals, is differentially employed to specify T-lineage fates in the thymus.