Intrathymic proliferation wave essential for V␣14؉ natural killer development depends on c-Myc

Marei Dosea, Barry P. Sleckmanb, Jin Hanc, Andrea L. Bredemeyerb, Albert Bendelacc, and Fotini Gounaria,1

aCommittee on , Department of Medicine, Rheumatology, and cHoward Hughes Medical Institute, Committee on Immunology, Department of Pathology, University of Chicago, Chicago, IL 60637; and bDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Harvey Cantor, Dana–Farber Institute, Boston, MA, and approved April 2, 2009 (received for review December 2, 2008) The molecular requirements for invariant V␣14-bearing natural the 100-fold more frequent CD44low stage 1 (ST1), they expand killer T cells (iNKT) in the thymus are poorly understood. A minute in numbers and down-regulate CD24. Both ST1 and the subse- Ϫ population of Ϸ500 newly selected CD69؉CD24؉ stage 0 (ST0) iNKT quent CD44high stage 2 (ST2) are NK1.1 . Most recent thymic ϩ cells gives rise to Ϸ100 times more CD44neg/loCD24؊ stage 1 (ST1) emigrants resemble CD4 ST2 cells and up-regulate NK cell cells, which then generate similar frequencies of CD44hiCD24؊ markers only as they mature in the periphery (9, 10), whereas an high ϩ stage 2 (ST2) and mature iNKT cells. Although the increased independent set of long-lived CD44 NK1.1 stage 3 (ST3) cells develop in the thymus (11). Apart from NK1.1, DX5 that number of ST1 compared with ST0 cells indicates the initiation of ␣ a proliferation wave in the very early stages of iNKT cell develop- detects an of 2 integrin (CD49b) can be used in conjunction with CD1d tetramers to identify later stage iNKT ment, details about the controlling mechanism are currently lack- ϩ ϩ cells, although NK1.1 and DX5 populations are not identical ing. Here, we show that the transcription factor c-Myc is required (12, 13). iNKT cell stages display differences in mRNA for iNKT cell development. Conditional ablation of c-Myc in double- expression and cytokine release on stimulation. Although ST1 positive specifically impacted iNKT but not conven- cells produce predominantly IL-4, subsequent stages also acquire tional T cell development. Within the iNKT population, a progres- the capability to produce IFN-␥ as they mature, as deduced from sive reduction of iNKT cells was observed starting at ST1 (Ϸ50-fold) in vitro receptor stimulation experiments and cytokine reporter and ST2 (Ϸ350-fold), with a complete lack of mature cells in knockin mice (1, 14). Two recent reports describe the zinc finger thymus, spleen, and liver. ST0/ST1 c-Myc-deficient iNKT cells transcription factor Plzf as an essential regulator of the iNKT cell IMMUNOLOGY showed reduced proliferation. In contrast, annexin V staining did effector phenotype (15, 16). Several other transcription factors not reveal increased apoptosis, and transgenic overexpression of have been implicated in iNKT cell development and function BCL-2 did not rescue iNKT cell development in c-Myc-deficient mice. (17). However, despite strong evidence indicating that the low Moreover, expression of known iNKT differentiation factors such infrequent CD44 iNKT precursors undergo massive expan- as Plzf and Gata3 was not dramatically altered. These, findings sion as they develop in the thymus, the factors that control cell provide compelling evidence that c-Myc mediates an intrathymic cycling and their overall impact on iNKT cell development remain unknown. proliferation wave immediately after agonist selection of iNKT cells The basic region/helix–loop–helix/leucine zipper (bHLHZip) and illustrate the importance of this expansion for the generation transcription factor myelocytomatosis oncogene (c-Myc) plays of mature iNKT cells in vivo. an integral role in proliferation, survival, and differentiation of normal and neoplastic cells. Myc binds E-box DNA motifs as a iNKT cells ͉ cell cycle ͉ nonconventional heterodimer with Max, resulting in cell cycle entry (18) and transcriptional activation or suppression of genes. c-Myc has atural killer T cells develop in the thymus and are charac- been implicated in cell proliferation (19) as well as the control Nterized by the expression of various surface molecules of cell growth. Its expression increases rapidly in response to originally detected on natural killer (NK) cells (1). The majority growth factors, receptor or TCR ligation, and conventional of murine NKT cells, referred to as iNKT, express a semiinvari- CD4 T cells expressing hypomorphic c-Myc alleles display pro- ant T cell receptor (TCR) repertoire with invariant V␣14-J␣18 found defects in activation induced proliferation (20). We and ␤ ␤ ␤ others have shown that c-Myc is essential for development at the usage in combination with V 8, V 7, or V 2 (2). Most exoge- pre-TCR checkpoint (21, 22). c-Myc has also been reported to nous ligands for NKT cells identified so far are components of control the self-renewal of hematopoietic stem cells (HSCs) (23), the cell wall of Gram-negative bacteria, implying an important and its conditional ablation in the bone marrow favored self- role in innate , but they also display autoreactivity and renewal over differentiation of HSCs in the stem cell niche (24). have been implicated in and cancer (re- Here, we show that c-Myc is essential for iNKT cell develop- viewed in ref. 1). ment. Conditional ablation of c-Myc at the DP stage in mice iNKT cells are positively selected on CD1d, an MHCI-like leads to a dramatic reduction of iNKT cells. CD44low Myc- molecule expressed on cortical CD4ϩCD8ϩ [double-positive deficient iNKT cells are not prone to apoptosis but proliferate (DP)] thymocytes in an agonist selection process involving less, providing evidence that c-Myc is involved in controlling the endogenous ligands, including the glycosphingolipid isoglobotri- proliferation wave of early iNKT cell development. hexosyl ceramide (iGb3) (3–5). iNKT cell selection is profoundly Results different from positive selection of conventional T cells, and coreceptor interactions are likely involved in the process. As an Myc Deficiency Disproportionately Impairs iNKT Cell Development. We have previously reported a strict requirement for the tran- example, homophilic interactions of SLAM and Ly108 surface scription factor c-Myc in the expansion of thymocytes undergo- molecules between iNKT and DP thymocytes induce activation of the Src kinase FynT leading to expansion and differentiation of immature iNKT cells (1). Author contributions: M.D., A.B., and F.G. designed research; M.D. and J.H. performed The development of CD1d tetramers loaded with ␣-galactosyl research; B.P.S. and A.L.B. contributed new reagents/analytic tools; M.D. analyzed data; ceramide (␣-GalCer) (6, 7) as a tool to label iNKT cells allowed and M.D. and F.G. wrote the paper. the identification of an extremely infrequent (Ϸ1/106) stage 0 The authors declare no conflict of interest. ϩ (ST0) CD24hiCD69 precursor population thought to represent This article is a PNAS Direct Submission. cells immediately after positive selection (8). As cells progress to 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812255106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Myc deficiency disproportionately impairs iNKT cell development. BALB/c control and CD4Cre Mycfl/fl mice were analyzed at 4–8 weeks of age. (A) (Upper) Representative FACS plots depicting CD4 and CD8 surface expression on lymphocytes from thymus and spleen. (B)(Upper) Invariant iNKT cell populations in thymus, spleen, and liver. (A and B)(Lower) Absolute cell numbers are given as histograms. N ϭ number of experimental mice per genotype indicated here. B, BALB/c; M, CD4Cre Mycfl/fl.(C) Quantitative PCR for Myc mRNA expression was performed on cDNA obtained from the indicated cell populations. No Myc expression was detected in CD4 SP or iNKT ST1 cells from CD4Cre Mycfl/fl mice. (D) iNKT cells from sublethally irradiated bone marrow chimeras were MACS enriched and analyzed by FACS. Host, Thy1.1ϩ BALB/c mice; donor, Thy1.2ϩ CD4Cre Mycfl/fl mice. Analysis was performed 12 weeks after injection. N ϭ 4.

ing ␤-selection (21). Here, we show that, although CD4Cre thymus (3.7 Ϯ 0.9 ϫ 105 vs. 9.9 Ϯ 3.2 ϫ 105) and spleen (1.0 Ϯ mediated deletion of c-Myc after ␤-selection has no significant 0.4 ϫ 106 vs. 2.8 Ϯ 0.8 ϫ 106) (Fig. 1B). effect on development at large, it specifically prohib- Thus, c-Myc ablation at the DP stage specifically and pro- its the development of iNKT cells (Fig. 1). Cellularity and foundly affected iNKT cells. To examine whether this defect was surface expression of CD4 and CD8 on thymocytes and spleno- cell intrinsic or due to altered properties of c-Myc-deficient DP Ϯ ϫ cells, which are essential for iNKT selection, we also analyzed cytes of BALB/c control and c-Myc-deficient thymi (2.1 0.6 ϩ 108 vs. 1.3 Ϯ 0.4 ϫ 108) and spleens (2.2 Ϯ 0.5 ϫ 108 vs. 2.3 Ϯ sublethally irradiated Thy1.1 BALB/c mice reconstituted with ϩ fl/fl 0.7 ϫ 108) were comparable (Fig. 1A). An up to 50% reduction a 1:1 mixture of host and Thy1.2 CD4Cre Myc donor bone in thymic cellularity with a corresponding reduction in DP cells marrow. These chimeras showed a disproportionate reduction of fl/fl could sometimes be observed but was not statistically significant iNKT cells originating from CD4Cre Myc donors. Thymocyte (1.7 Ϯ 0.5 ϫ 108 vs. 1.0 Ϯ 0.3 ϫ 108). Likewise, CD4Cre Mycfl/fl preparations were enriched for iNKT cells with PBS57 loaded mice tended to have fewer peripheral T cells, but only the CD1d-tetramers (hereafter referred to as tetramer) using mag- netic microbeads. The ratio of tetramerϩ to tetramerϪ host- reduction in CD4 single-positive (SP) cells was statistically Ϫ 8 8 derived (Thy1.2 ) cells was 1:4, whereas it was 1:180 in donor- significant (4.1 Ϯ 1.3 ϫ 10 vs. 1.8 Ϯ 0.8 ϫ 10 ). Thus, with the ϩ derived (Thy1.2 ) cells (Fig. 1D), suggesting that iNKT cell exception of mild defects, CD4Cre Mycfl/fl mice displayed normal development is controlled by c-Myc in a cell-intrinsic fashion. distribution of conventional T cell subsets in thymus and spleen. To our surprise, however, we found an almost complete c-Myc Expression Is Required During iNKT Cell Development. To ablation of iNKT cells in thymus, spleen, and liver (Fig. 1B). measure c-Myc protein levels in the different stages of iNKT cell Absolute numbers of iNKT cells were reduced almost 70-fold development, we analyzed MycG/G mice (25). These knockin Ϯ ϫ 6 Ϯ ϫ 6 in the thymus (1.3 0.5 10 vs. 0.020 0.015 10 ) and mice express an N-terminal c-Myc-GFP fusion protein and thus 30-fold in the spleen (1.6 Ϯ 0.4 ϫ 106 vs. 0.05 Ϯ 0.03 ϫ 106), allow the detection of c-Myc protein expression by flow cytom- where they reached the limit of detection. Myc mRNA in etry. iNKT cells were MACS enriched from pools of control and CD4Cre Mycfl/fl mice was below detection limits in both CD4 MycG/G mice by using tetramers and magnetic beads, and stained SP as well as the few remaining iNKT cells, indicating efficient with B220, heat-stable (HSA), CD44, and DX5 anti- deletion (Fig. 1C). We also looked at ␥␦-T cells as another bodies to discriminate the developmental stages 1–3 (Fig. 2A). small population and found increased numbers in Thymocyte suspensions were also stained for CD4, CD8, and

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812255106 Dose et al. Downloaded by guest on September 24, 2021 Fig. 2. c-Myc expression in developing iNKT cells. (A) Flow cytometric analyses of Myc-GFP knockin mice. Overlay histograms show GFP expression compared to the corresponding population of control mice. Populations were gated as indicated. For iNKT cell subsets, thymocytes from 3 mice were pooled and magnetically enriched for CD1d-tetramer-positive cells before FACS analysis. Numbers indicate percentages of cells with intermediate and high levels of c-Myc-GFP in MycG/G mice. The experiment was repeated 3 times with 3 mice in each group, or pools of 3 mice for MACS enrichment. (B) Overlay histogram of c-Myc-GFP expression in iNKT stages 1–3 from MycG/G mice. (C) Quantitative RT-PCR was performed on cDNA obtained from sorted cells as indicated. IMMUNOLOGY TCR␤. As reported, c-Myc expression was highest in double- regulated in the DPhi, CD4ϩ SP, and iNKT ST1, -2, and -3 stages negative (DN) cells, containing the highly proliferating DN2 and when compared to DPlo cells (Fig. 2C). In summary, these data DN4 cells. TCR␤Ϫ DP (DPlo) cells, mostly preselected DP cells, support a role for c-Myc during iNKT development, potentially had the least amount of Myc-GFP, whereas Ϸ4% of cells on selection. expressed intermediate levels of c-Myc in TCR␤high DP (DPhi) To determine the precise stage of iNKT cell development and CD4 SP cells, indicating that these populations contain a affected by c-Myc ablation, MACS-enriched iNKT cells from small percentage of dividing cells. Interestingly, 46% of the ST1 BALB/c and CD4Cre Mycfl/fl mice were stained for HSA, CD44, and Ϸ32% of the ST2 and ST3 iNKT cells expressed interme- and DX5 surface expression. Myc deficiency resulted in a diate levels of c-Myc-GFP. c-Myc expression levels were highest complete loss of ST3 and a severe loss of ST1 and ST2 iNKT in ST1 cells (Fig. 2B), indicating that c-Myc might be up- cells. More than 80% of tetramerϩHSAϪ c-Myc-deficient iNKT regulated on selection to initiate a proliferative burst and then cells were ST1 cells compared with Ϸ30% in control mice (Fig. stabilize at intermediate levels. Notably and consistent with the 3A). Whereas ST3 cells were absent, ST2 cellularity was reduced proliferative burst required to expand the iNKT cell compart- Ϸ350-fold (44,000 vs. 150 event counts when acquiring all cells ment on selection, the percentages of c-Myc-GFP intermediate- enriched from 1 thymus) (Fig. 3B). ST1 cells were reduced in expressing cells are considerably higher in postselected iNKT numbers Ͼ50-fold (44,000 vs. 700), whereas ST0 cells were cells than in postselected DPhi thymocytes. present at comparable numbers (500 vs. 700), indicating that In agreement with the protein levels, Myc mRNA was up- these cells are less affected by c-Myc ablation, presumably

Fig. 3. c-Myc deficiency severely reduces iNKT ST1, ST2, and ST3 cells. (A)(Upper) FACS plots after magnetic bead enrichment for tetramerϩ cells. Numbers on plots indicate total event counts. (Lower) MACS-enriched tetramerϩHSAϪ cells are plotted for CD44 vs. DX5 surface expression to discern iNKT ST1 to -3. Numbers indicate percentages. (B) Total event counts in gate corresponding to acquisition of 1 thymus. Results are representative of 2 experiments with a total of 6 mice per group from 2 different litters.

Dose et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 Fig. 4. Reduced proliferation in c-Myc-deficient iNKT cells. Mice were injected with EdU and analyzed 3 h later. Totals of 2–3 BALB/c and 3–5 CD4Cre Mycfl/fl thymi were pooled and MACS enriched for iNKT cells. Events were gated as indicated. The experiment was performed with 3 independent pools of mice. Numbers under EdU histograms are EdUϩ events/total events in population.

Fig. 5. c-Myc deficiency does not impair iNKT survival. (A) FACS analyses of because they are just upstream of a proliferative burst. Alter- tetramer-enriched thymocytes from pools of 4-week-old CD4Cre Mycfl/fl and fl/fl natively, despite the absence of Myc mRNA in iNKT ST1 Myc littermates. Events were gated as indicated. SSC, side scatter. (B) FACS fl/fl analyses of thymocytes from littermates of the indicated genotypes. Results thymocytes from CD4Cre Myc animals (Fig. 1C), it is possible are representative of 3 independently analyzed litters. that some cells escape timely Cre-mediated deletion or retain c-Myc protein for a limited time. In conclusion, c-Myc ablation severely reduces the cellularity of all immature iNKT cells, with fraction of annexin Vϩ cells was Ϸ2% in ST1 iNKT cells in both the exception of the earliest ST0 further supporting a role for cases (Fig. 5A), indicating that c-Myc deficiency does not lead to c-Myc on selection of iNKT cells. excessive cell death. annexin V staining may, however, fail to identify apoptotic processes that nevertheless occur in vivo. In an Loss of Proliferating ST0/ST1 iNKT Cells on c-Myc Deletion. c-Myc is independent approach, we therefore crossed CD4Cre Mycfl/fl an important mediator of cell proliferation and the loss of c-Myc mice with the vav-bcl-2 transgenic strain. These mice overexpress in lymphocytes impairs their capacity to cycle (20, 21). In human BCL-2 in all hematopoietic cells under the control of the addition, in vivo BrdU uptake experiments indicated that early vav promoter (26). FACS analysis of thymus and spleen from stage iNKT cells actively divide, whereas thymic stage 3 cells do CD4Cre Mycfl/fl vav-bcl-2 mice showed that transgenic expression not (9). To address whether c-Myc deficiency affected the of BCL-2 could not ameliorate the iNKT cell deficiency of capacity of immature iNKT cells to cycle, we injected the CD4Cre Mycfl/fl mice (Fig. 5B). In conclusion, we did not observe thymidine analog 5-ethynyl-2Ј-deoxyuridine (EdU) into CD4Cre excessive cell death in c-Myc-deficient iNKT cells nor could the Mycfl/fl and BALB/c control mice. Three hours later, 2–3 control loss of iNKT cells on c-Myc deletion be recovered by enforced or 3–5 CD4Cre Mycfl/fl thymi were pooled and magnetically survival through transgenic BCL-2 expression. Thus, we did not enriched with tetramers and MACS beads to obtain large enough cell numbers for analysis. We observed a Ͼ10-fold find evidence that Myc deficiency predisposes developing iNKT reduction (10.4 Ϯ 1.4-fold) in EdUϩ cells on loss of c-Myc in the cells to apoptosis. ST0/1 compartment (B220ϪCD44lowDX5Ϫ) (Fig. 4). Thus, im- mature CD44low iNKT cells depend on c-Myc for proliferation, Expression of Lineage Determinants. To investigate whether lack of and their impaired cycling capacity preventing exponential ex- c-Myc altered the expression of developmental regulators, we pansion might explain the lack of the subsequent developmental performed quantitative real-time RT-PCR using TaqMan iNKT cell stages in CD4Cre Mycfl/fl mice. We also observed a probes (Fig. 6). Given the proliferative defect we observed in 2.5-fold reduction in proliferating c-Myc-deficient ST2 c-Myc-deficient iNKT cells, we measured mRNA levels of cyclin (CD44highDX5Ϫ) cells; however, this was not statistically signif- D2, a downstream target that can also be indirectly regulated by Ϯ c-Myc. Cyclin D2 expression was 50% lower in c-Myc-deficient icant (2.5 1.5-fold) probably because of the extremely low ϩ number of events in this population (e.g., 281 events from pools CD4 SP thymocytes and ST0 iNKT cells compared with of 5 mice). controls, but it was comparable or even slightly elevated in ST1 cells. The proliferative effect of c-Myc may therefore be inde- No Excessive Cell Death on c-Myc Deletion. Apart from its critical pendent of cyclin D2. role in mediating proliferation, c-Myc has also been implicated Thus far, our observations have linked impaired iNKT cell in controlling survival in several systems. Thus, the loss of iNKT development in c-Myc-deficient thymocytes to reduced prolif- cells on ablation of c-Myc may also be caused by increased cell eration in immature iNKT subsets, whereas there was no evi- death. We performed annexin V staining of MACS-enriched dence for increased apoptosis. Apart from impacting prolifera- thymocytes from CD4Cre Mycfl/fl and Mycfl/fl littermates. The tion and apoptosis, however, c-Myc deficiency could also alter

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812255106 Dose et al. Downloaded by guest on September 24, 2021 obscured by the general lack of mature iNKT cells because of the lack of proliferative expansion. Discussion Here, we provide evidence that Myc ablation in DP thymocytes specifically impacts iNKT cell development but not the devel- opment of conventional T cells. In contrast to developing conventional T cells, intrathymic iNKT cell differentiation de- pends on agonist selection as well as costimulation (29). iNKT cells are not killed by negative selection on encounter of agonist but rather the receptor signal they receive places them in a ‘‘standby’’ mode, priming them for immediate response to further receptor challenge (1, 7). Selection and development of iNKT cells in the thymus have been proposed to follow a program that resembles T cell activation rather than the devel- opment of conventional T cells (14). This includes a mechanis- tically elusive proliferation wave after selection to account for the substantial expansion observed between ST0 and ST1 of thymic iNKT cell development (7). Our finding that c-Myc ablation reduces proliferation of ST0/ST1 cells shows that this transcription factor is a crucial mediator of proliferation in immature iNKT cells. The observation that this leads to a complete lack of mature iNKT cells highlights the significance of proliferation in iNKT cell development. c-Myc has been impli- cated in proliferative processes along multiple stages of hema- Fig. 6. Expression of lineage determinants. cDNA from 2,000 cells per topoietic development. Among these processes are the self- population was subjected to quantitative PCR. ST0 and ST1 cells were sorted renewal of HSCs (24), the activation of peripheral CD4 T cells after MACS enrichment. DPhi and CD4 SP cells were sorted from the tetramer- after TCR stimulation (20), as well as the proliferative burst after IMMUNOLOGY depleted fraction. The experiment was repeated 3 times with comparable results. pre-TCR assembly and signaling. A mechanism for c-Myc induction in response to TCR stim- ulation has been proposed by analyzing c-Rel and RelA double- the expression of lineage determinants controlling the differen- deficient T cells. It was shown that growth and proliferation of activated T cells requires induction of c-Myc by Rel/NF-␬B tiation of iNKT cells. To examine whether there was a deregu- ␪ ␪ lation of critical differentiation factors, we measured mRNA activity. This process depends on protein kinase C (PKC )- levels of factors previously implicated in T cell differentiation. controlled nuclear translocation of c-Rel and AP-1/NFAT- induced transcription of RelA (30). Based on the resemblance These included the zinc-finger domain containing transcription between iNKT cell development and activation of conventional factors Plzf and Th-POK (c-Krox) as well as Gata3. cDNA was T cells, it is tempting to speculate that this mechanism is also obtained from 2,000 sorted cells from pools of 4 mice each. The involved in iNKT development. Both ablation of PKC␪ (31) and experiment was repeated 3 times with similar results (Fig. 6). All inhibition of AP-1 signaling (32, 33) resulted in loss of iNKT experiments showed clearly detectable Plzf expression that was cells; however, the observed phenotypes in these studies are less higher in ST1 than in ST0 iNKT cells. Therefore, c-Myc ablation severe and affect later developmental stages compared with does not appear to prevent up-regulation of Plzf. The expression c-Myc ablation. This in turn would indicate that c-Myc is of Th-POK, which is essential for CD4 T cell differentiation (27) required before NF-␬B and AP-1 signaling at an earlier stage in and also expressed in iNKT cells (16), was unperturbed by Myc iNKT cell development. deletion. Gata3, which has been implicated in the development Although c-Myc clearly controls proliferation during iNKT of virtually all lymphocyte lineages (28) was down-regulated in cell development, we did not observe excessive cell death in c-Myc-deficient DPhi thymocytes but unchanged in CD4 SP and Myc-deficient iNKT cells nor did transgenic expression of BCL-2 slightly elevated in ST0 and ST1 iNKT cells. Thus, critical rescue the phenotype of CD4Cre Mycfl/fl mice, arguing against a differentiation factors are expressed in c-Myc-deficient iNKT role for c-Myc in apoptosis of developing iNKT cells. Likewise, cells at comparable or slightly altered levels. we did not detect severe changes in the expression of transcrip- Early-stage iNKT cells express higher levels of IL-4 mRNA tion factors previously implicated in iNKT cell development. than later stages and respond predominantly by IL-4 production Both Plzf, a critical factor for iNKT cell function, and Th-POK, when stimulated (9). Compared with control cells, c-Myc- a master regulator of CD4 T cell development, are detectable in deficient iNKT ST0 and ST1 cells expressed lower amounts of c-Myc-deficient ST0 and ST1 cells, whereas Gata3 appears IL-4 mRNA, indicating that these cells may not be differentiated slightly elevated. Even small changes in the levels of transcription factors may deregulate networks in control of developmental sufficiently to up-regulate IL-4 to wild-type levels or that c-Myc processes (34). It remains to be determined whether the small is required for proper control of IL-4 expression. Unfortunately, fl/fl transcriptional alterations we observed reflect a significant the extremely low number of thymic CD4Cre Myc iNKT cells disturbance in the transcriptional network controlling iNKT cell precludes further analysis of potential defects in cytokine pro- development. duction on stimulation as well as broader screening approaches In conclusion, data presented here provide mechanistic depth for differentially regulated genes. for our understanding of the early developmental stages of the At the current state of the art, our data do not support a enigmatic iNKT cell lineage. Our data provide compelling specific role for c-Myc in controlling iNKT signature. However, evidence that c-Myc mediates an intrathymic proliferation wave we cannot formally rule out that c-Myc ablation affects differ- immediately after agonist selection of iNKT cells and illustrate entiation as well as proliferation. In addition, effects on differ- the importance of this expansion for the generation of mature entiation may be difficult to dissect because they might be iNKT cells in vivo.

Dose et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 24, 2021 Materials and Methods labeling kit (BD Pharmingen) was used according to the manufacturer’s Mice. All mice were kept in the animal facilities of the University of Chicago instructions. All samples were analyzed on an LSRII or sorted on a FACSAria according to protocol no. 71880 approved by the Institutional Animal Care instrument (BD Biosciences). Data were analyzed by using FlowJo software and Use Committee. Mycfl/fl mice (35) were crossed with CD4Cre transgenic (Tree Star). mice (36). CD4Cre Mycfl/wt mice were backcrossed to the BALB/c background for 6 generations and then crossed inter se to obtain CD4Cre Mycfl/fl animals. Irradiation Bone Marrow Chimeras. Sublethally irradiated (650 rad; Gammacell ϩ BALB/c control mice were purchased from Charles River. The CD4Cre trans- 40) Thy1.1 BALB/c mice (host) were injected with a 1:1 mixture of FACS-sorted ϩ fl/fl ␤ gene was detected by PCR using 5Ј-ATCGCTCGACCAGTTTAGT-3Ј (forward) host and Thy1.2 CD4Cre Myc donor lineage (B220, CD19, TCR , CD8, Gr-1, ϫ 5 and 5Ј-CGATGCAACGAGTGATGA-3Ј (reverse), and the floxed Myc allele was Mac-1, DX5) negative bone marrow (2 10 cells per mouse). Animals were detected with 5Ј-TAAGAAGTTGCTATTTTGGC-3Ј (forward) and 5Ј-TTTTCTTTC- treated with Bactrim in the drinking water for the entire time of observation CGATTGCTGAC-3Ј (reverse) primers. vav-bcl2-Tg mice were a gift from A. T. (12 weeks). Look (Dana–Farber Cancer Institute, Boston, MA) and were originally made by J. Adams and colleagues (26). MycG/G mice were provided by B. P. Sleckman Cell Cycle Analysis. Mice were injected intravenously with 1 mg of EdU (Washington University, St. Louis, MO). (Invitrogen) 3 h before analysis. Thymocytes were processed and stained for flow cytometry according to the manufacturer’s instructions. Flow Cytometry. Multicolor-FACS stainings were performed for analysis and cell sorting of primary thymocytes. A total of 1–5 ϫ 106 cells were stained in a Quantitative Real-Time PCR. A total of 2,000 cells were sorted directly into lysis total volume of 50–200 ␮L of FACS buffer (HBSS/2% FBS/50 ␮g/mL DNase I). buffer, and RNA was extracted by using the RNeasy Micro kit (Qiagen). cDNA were from BD Pharmingen or eBioscience: B220-PacificBlue(RA3- was prepared with the SuperScript-III RT kit (Invitrogen). Quantitative PCR was 6B2), CD4-FITC/-peridinin chlorophyll protein (PerCp)-Cy5.5/-allophycocyanin performed on an ABI7300 machine (Applied Biosystems). All targets were ␤ (APC)-Alexa750/-phycoerythrin (PE)-Cy7(RM4-5), -PE/-APC(GK1.5), CD8-FITC/- determined relative to -actin expression by using TaqMan PerCp-Cy5.5/-APC-Alexa750/-PE/-APC/-PE-Cy7/-PacificBlue(53-6.7), TCR␤-PE/- Assays from Applied Biosystems. Data were analyzed and evaluated according ⌬⌬ APC/-APC-Alexa750(H57-597), TCR␥␦-PE/-FITC(eBioGL3), pan-NK-FITC(DX5), to the relative CT method. CD44-PerCp-Cy5.5(IM7), CD69-FITC/-PE(H1.2F3), CD24-PE(M1/69). For analysis of iNKT cells, thymocyte single-cell suspensions from 1–10 mice were stained ACKNOWLEDGMENTS. We thank A. Savage for generously sharing technical for1honicein500␮L of HBSS/2% FCS with APC-labeled CD1d tetramers expertise, advice, and for helpful discussions, and L. Molinero for help with injections. We are grateful to M. Morrin for excellent technical assistance and loaded with the ␣-GalCer analog PBS57 (37) obtained from the tetramer core to R. Duggan, D. Leclerc, M. Olson, and J. Cao for expert assistance with cell facility of the National Institutes of Health. Tetramer-positive cells were then sorting. We thank the members of the Gounari and Bendelac Laboratories for enriched with anti-APC magnetic microbeads by using an autoMACS cell their support. This work was funded by National Institutes of Health Grant R01 separator (Miltenyi Biotec) as described in ref. 8 and subjected to further AI059676 (F.G.). A.B. is an investigator of the Howard Hughes Medical Insti- staining for flow cytometry. To detect apoptotic cells, the annexin V-FITC tute. M.D. is supported by the Lady Tata Memorial Trust.

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