ARTICLE Diff erential synergy of Notch and T cell receptor signaling determines αβ versus γδ lineage fate Annette I. Garbe,1 Andreas Krueger,1 Fotini Gounari,1,2 Juan Carlos Zúñiga-Pfl ücker,3 and Harald von Boehmer1 1Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 2Tufts-New England Medical Center, Boston, MA 02111 3University of Toronto, Toronto, Ontario, M4N 3M5 Canada Thymic precursors expressing the pre–T cell receptor (TCR), the TCR, or the TCR can all enter the CD4+8+ lineage, albeit with different effi cacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still signifi cantly contributes to the generation of T cells. In particular, in lineage commitment, the pre-TCR synergizes more effi ciently with Notch signals than the other two TCRs, whereas TCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of versus lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the effi cacy of T cell generation. CORRESPONDENCE The mammalian immune system contains sev- essential for the rescue of DP cells from pro- Harald von Boehmer: eral T lymphocyte lineages of which the grammed cell death, which requires binding Harald_von_Boehmer@ αβTCR lineage and the γδTCR lineage di- with relatively low avidity to intrathymic dfci.harvard.edu verge relatively early in T lymphocyte devel- MHC ligands and results in the generation of + + + + Abbreviations used: DL-1, opment, resulting in CD4 /8 cells expressing CD4 helper and CD8 cytolytic T cells de- Delta-like 1; DN, double nega- an αβTCR and CD4−8− double negative (DN) pending on the binding of the αβTCR to class tive; DP, double positive; GSI, γδ γ-secretase inhibitor; pTα, cells with a TCR. II and class I MHC molecules, respectively pre-TCRα. The gene segments encoding the γδTCR (8–10). This binding also results in termination rearrange slightly before or at the same time as of Ptcra gene transcription (11). The Journal of Experimental Medicine the Tcrb v-gene segments that encode the Although the above scenario represents the TCRβ chain. T cells expressing the αβ T cell most eff ective pathway of generating αβ lin- receptor develop from precursors that fi rst re- eage cells, it does not represent the only one. arrange the Tcrb locus (1, 2) and express TCRβ In Ptcra knockout mice, it was found that not chains in association with the invariant pre- only the pre-TCR but also γδTCRs or some TCRα (pTα) chain (3, 4). The pre-TCR is prematurely expressed αβTCRs in DN3 cells required for the rescue of CD25+44− (DN3) could rescue the generation of bona fi de αβ cells from programmed cell death (5) and is es- lineage cells (12): Ptcra−/− Tcrd−/− mice are un- sential for extensive proliferation of the rescued able to make either the pre-TCR or the cells that eventually become CD4+CD8+ dou- γδTCR. In these mice, it was shown that pre- ble positive (DP) cells that rearrange the Tcra maturely expressed αβTCRs could rescue αβ locus (6). TCRα chains when compared with T cell development by facilitating the genera- the pTα chain compete favorably for covalent tion of DP cells that all contained productively pairing with the TCRβ chain (7), resulting in rearranged TCRβ chains (12). In Ptcra−/− expression of the αβTCR. The αβTCR is Tcra−/− mice, γδTCRs could rescue the devel- opment of αβ lineage DP cells of which only A.I. Garbe and A. Krueger contributed equally to this work. 15% expressed TCRβ chains in the cytoplasm The online version of this article contains supplemental material. (12). The latter observation is well in line with JEM © The Rockefeller University Press $8.00 1579 Vol. 203, No. 6, June 12, 2006 1579–1590 www.jem.org/cgi/doi/10.1084/jem.20060474 data showing that in pre-TCR–defi cient mice, such as mitment (16), whereas elimination of the Notch-dependent Ptcra−/− and Tcrb−/− mice, γδTCR-expressing cells can enter transcription factor CSL (RBP-Jκ) at a later stage was asso- the αβ lineage, abolish γδTCR expression, and rearrange ciated with impairment of αβ lineage development (17). the Tcra locus (12, 13). However, only a fraction of (DN) Again, the latter in vivo experiments did not establish cells with αβTCRs or γδTCRs become αβ lineage DP cells whether Notch contributed to the survival and/or prolifer- because many of the DN cells expressing a γδTCR or ation of cells after lineage commitment or whether it was αβTCR become functionally mature CD4−8− cells in which directly involved in lineage commitment. This question TCR ligation results in cytokine secretion (13, 14). Such was analyzed in more detail in a recently developed culture functionally mature CD4−8− cells, however, are not found system, which revealed that Notch receptor Delta-like 1 among pre-TCR–expressing cells. These experiments sug- (DL-1) ligands were required for early T lineage commit- gest that the type of TCR expressed in immature precursors ment as well as diff erentiation of CD44+25− (DN1) cells can contribute to lineage choice but that in all likelihood into CD44+25+ (DN2) and CD25+44− (DN3) cells, repre- other factors have a decisive role as well. Here we have asked senting sequential steps of development before TCR ex- in which way can DN3 precursor cells that do not express the pression (18). Also, at the DN3 stage, pre-TCR–expressing pre-TCR, i.e., DN3 cells with γδTCRs or αβTCRs, enter cells required Notch ligands to further develop into αβ the αβ lineage. lineage cells (19). By analysis of DN3 cells from RAG- In the context of these questions, it is of interest that defi cient mice that are unable to rearrange TCR loci, it was experiments addressing the role of Notch in αβ versus γδ T found that DL-1 ligands contributed a survival signal at this cell lineage decision concluded that higher levels of Notch1 checkpoint (20). receptors on T cell precursors favored the development of Here we have investigated whether Notch contributes to αβ lineage over γδ lineage cells under conditions where the entry into the αβ lineage of pre-TCR–, γδTCR-, and Notch1+/+ cells were competing with Notch1+/− precursors αβTCR-expressing precursors at the DN3 stage of develop- and in which more αβ lineage cells had the Notch1+/+ than ment and whether there is diff erential synergy of Notch sig- the Notch1+/− genotype (15). It was not clear from these nals with signals generated by diff erent TCRs. To this end, data whether Notch favored survival and/or proliferation of we cultured DN3 cells from mice that can express various committed αβ lineage precursors or whether Notch signal- TCRs in the presence or absence of Notch DL-1 ligands and ing was required for αβ lineage choice. Subsequent studies have quantitated the requirement for Notch signals by in- concerning the role of Notch in T cell development using hibiting Notch signaling with a γ-secretase inhibitor (GSI). knockout strategies revealed that Notch signaling played an Furthermore, we analyzed whether Notch signaling contributes essential role at an earlier stage in T versus B lineage com- to the proliferation and diff erentiation of cells at the DN4 Figure 1. Phenotype of mice with different TCRs. Thymocytes from TCRγδ transgenic mice on the Rag-1−/− background (TCRγδ), and lck-pTα transgenic mice on the Ptcra−/− background (lck-pTα), Ptcra−/− RAG-1−/− mice (Rag−/−) were stained with CD4, CD8, TCRγδ, and TCRβ mice (pTα−/−), lck-TCRα mice on the Ptcra−/− background (lck-TCRα), antibodies. Numbers in each quadrant indicate the percentage of cells. 1580 NOTCH AND TCR SIGNALING IN LINEAGE COMMITMENT | Garbe et al. ARTICLE stage of development. The fi ndings suggest a novel model of timing of receptor expression is equivalent to that seen in αβ T cell lineage commitment in which the extent of syn- normal mice. The γδTCR transgenic mice express the ergy between Notch signaling and TCR signaling determines transgenic TCR at the DN4 stage of development as it oc- lineage fate and in which diff erent TCRs make a defi nite curs in normal mice. Fig. 1 shows the phenotypic analysis contribution to lineage choice. of thymuses from these mice. Introduction of the pTα chain under the lck promoter into Ptcra−/− mice results in RESULTS a normal thymic phenotype, with DP cells representing Phenotype of mice with different TCRs the vast majority of cells and a minor fraction of DN To study the impact of diff erent TCRs on lineage devel- γδTCR- or αβTCR-expressing cells (21). Ptcra−/− mice opment, we used the following mice: fi rst, lck-pTα trans- have few DP cells and mostly DN γδTCR- and fewer genic mice on the Ptcra−/− background that can mostly DN αβTCR-expressing cells. Lck-TCRα mice on the produce the pre-TCR as well as diverse γδTCRs and few Ptcra−/− background have a smaller proportion of DP cells diverse αβTCRs; second, Ptcra−/− mice that cannot make than lck-pTα mice and an increased proportion of DN the pre-TCR but do make diverse γδTCRs and a few cells expressing γδTCRs and αβTCRs. TCRγδ trans- αβTCRs by rearrangement of endogenous genes; third, genic Rag-1−/− mice have DP cells, some of which express lck-TCRα mice on the Ptcra−/− background that instead low levels of the transgenic TCR, and few DN cells with of the pre-TCR express diverse αβTCRs at an early stage low levels of the γδTCR when compared with γδ T cells of development, exaggerating the few DN cells that ex- from Ptcra−/− mice.