The T-box transcription factors T-bet and Eomesodermin (Eomes) instruct discrete stages in NK cell development. However, their role in the immune response of mature NK cells against pathogens remains unexplored. We used an inducible deletion system to elucidate the cell-intrinsic role of T-bet and Eomes in mature NK cells during the course of mouse CMV infection. We show both T-bet and Eomes to be necessary for the expansion of virus-specific NK cells, with T-bet upregulation induced by IL-12 signaling and STAT4 binding to a conserved enhancer region upstream of the Tbx21 loci. Interestingly, our data suggest maintenance of virus-specific memory NK cell numbers and phenotype was dependent on T-bet, but not Eomes. These findings uncover a nonredundant and stage-specific influence of T-box transcription factors in the antiviral NK cell response.
Although NK cells are innate lymphocytes, they are now appreciated to possess developmental and functional traits in common with T and B cells of adaptive immunity (1). In particular, a subset of NK cells expressing the activating Ly49H receptor, which can bind the mouse CMV (MCMV)-encoded glycoprotein m157, will undergo a clonal-like proliferation and generate long-lived memory cells (2) similar to Ag-specific CD8+ T cells encountering pathogen-derived peptides presented on MHC class I. Specific proinflammatory cytokines and transcription factors have been shown to promote adaptive NK cell responses in settings of infection and cancer (3). Specific defects in the clonal expansion of Ly49H+ NK cells have been shown to cripple host control of MCMV (3). Despite recent advances in defining the rapid clonal expansion of effector NK cells, the mechanisms governing the formation and maintenance of memory NK cells during viral infection remain to be elucidated.
T-box family transcription factors T-bet and Eomesodermin (Eomes) have wide-ranging effects that direct lymphocyte immunity. Although recent studies have documented the importance of T-bet and Eomes in NK cell development and function (4–8), their influence on pathogen-specific NK cell responses is unknown. Using an inducible deletion system where T-bet and Eomes can be individually deleted in mature NK cells, we have discovered a stage-specific and nonredundant role for T-box transcription factors during NK cell homeostasis, antiviral response, and generation of long-lived memory.
Materials and Methods
Mice, tamoxifen treatment, and MCMV infection
All of the mice used in this study were bred and maintained at the Memorial Sloan Kettering Cancer Center in accordance with Institutional Animal Care and Use Committee guidelines. Mixed bone marrow chimeric mice were generated, and adoptive transfer studies were performed as previously described (2). Mice were infected by i.p. injections of MCMV (Smith strain) with 7.5 × 103 PFUs. Mice were administered 8 mg tamoxifen dissolved in 200 μl olive oil by oral gavage on days 0, 1, and 3. Control mice received 200 μl olive oil.
Flow cytometry and cell sorting
Single-cell suspensions were prepared from indicated organs and Fc receptors were blocked with 2.4G2 mAb before staining with the indicated surface or intracellular Abs (BD Biosciences, BioLegend, or eBioscience). Flow cytometry was performed on an LSR II flow cytometer (BD Biosciences). Cell sorting was performed on an Aria II cytometer (BD Biosciences). All data were analyzed with FlowJo software (Tree Star). NK cell enrichment and adoptive transfers were performed as previously described (9).
ChIP and quantitative RT-PCR
Chromatin immunoprecipitation (ChIP) and quantitative RT-PCR were performed as described previously (10). The following quantitative RT-PCR primers were used for ChIP studies: CNS1 pTbx21, forward (For): 5′-CTAAGCAGGCACTCCATCAGTTG-3′, reverse (Rev): 5′-GTCCTTCCTCCGCTGTTCTATTC-3′; CNS2 pTbx21, For: 5′-TAGCGGAAAGCGAGATGGTG-3′, Rev: 5′-AGTGAAGGAGTTCTGTGGTTCTGG-3′; CNS3 pTbx21, For: 5′-GAGCCGACATACTGACATTCTGC-3′, Rev: 5′-CATTCTCCTCTCCCACCATCTTG-3′; Ifng promoter, For: 5′-GCTCTGTGGATGAGAAAT-3′, Rev: 5′-GCTCTGTGGATGAGAAAT-3′; Gene desert 50 kB upstream of Foxp3, For: 5′-TAGCCAGAAGCTGGAAAGAAGCCA-3′, Rev: 5′-TGATACCCTCCAGGTCCAACCATT-3′; Utf1, For: 5′-AGTCGTTGAATACCGCGTTGCTG-3′, Rev: 5′-CTGTTGAGATGTCGCCCAAGTGC-3′; Zpf42, For: 5′-AGAGGGCGGTGTGTACTGTGGTG-3′, Rev: 5′-CTTCTTCTTGCACCCGGCTTGAG-3′.
ChIP-seq, RNA-seq, and sequencing analysis
ChIP-seq, RNA-seq, and analysis of sequencing data were all performed, as previously described (11). The Gene Expression Omnibus accession numbers for the ChIP-seq and RNA-seq data reported in this paper are GSE106137 and GSE106138, and can be found in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/).
All plots depict mean ± SEM, except RNA-seq counts, which show mean ± SD. Two-tailed unpaired Student t test was used to derive statistical differences, unless otherwise indicated. A p value >0.05 was considered significant. Plots and statistical analyses were produced in GraphPad Prism or R (v 3.3.3).
Results and Discussion
T-box transcription factors are dispensable for NK cell homeostasis
To investigate the role of individual T-box transcription factors on the homeostasis and antiviral response of mature NK cells without impacting their development, we generated mice containing a floxed Eomes or T-bet (Tbx21) gene and a Cre recombinase-human estrogen receptor ligand-binding domain (ERT2) fusion protein expressed downstream of the ubiquitin promoter (Ub) (Eomesf/f × UbCre-ERT2 or Tbx21f/f × UbCre-ERT2 mice, respectively). Treatment of Eomesf/f × UbCre-ERT2 or Tbx21f/f × UbCre-ERT2 mice or cells with tamoxifen causes the specific excision of the floxed T-box transcription factor gene and loss of protein in cells of interest (Fig. 1A, data not shown). Cells where the genes encoding Eomes or Tbx21 are ablated following tamoxifen treatment will be referred to as Eomes−/− or T-bet−/−, respectively.
We first tested whether Eomes expression was required for the homeostasis of mature NK cells. We isolated purified NK cells (Supplemental Fig 1A, 1B) from the spleen of Eomesf/f × UbCre-ERT2 mice (CD45.2) and cotransferred them with an equal number of wild-type (WT) NK cells (CD45.1) into WT recipients (CD45.1 × CD45.2), which were immediately treated with a tamoxifen regimen to efficiently induce Eomes deletion. Greater than 1 wk following tamoxifen treatment, we found equal numbers of Eomes−/− and WT NK cells (Fig. 1B), suggesting Eomes is not necessary for the maintenance of total NK cell numbers under steady state conditions. However, Eomes−/− NK cells exhibited a slight increase in the percentage TRAIL+ cells and a decrease in the percentage Ly49H+ cells in both the spleen and liver (Supplemental Fig. 1C). Using CD27 and CD11b as markers of NK cell maturation (12), no significant changes were observed between Eomes−/− and WT splenic NK cells (Supplemental Fig. 1D).
When Tbx21f/f × UbCre-ERT2 NK cells were transferred with WT NK cells and recipients treated with tamoxifen, equal numbers of cotransferred populations were recovered 1 wk later as well (Fig. 1C), suggesting T-bet, like Eomes, is also not essential for the maintenance of mature NK cell numbers at steady state, and that perhaps T-box transcription factors compensate for the loss of one another during basal homeostasis. In contrast to Eomes deletion, NK cells that were deleted of T-bet exhibited a significant decrease in the percentage of TRAIL+ cells, and increase in the percentage of Ly49H+ NK cells in the spleen and liver (Supplemental Fig. 1E). A significant increase in the percentage of CD27+ CD11b+ cells was also observed (Supplemental Fig. 1F). In all experiments, a control set of mice receiving oil alone showed no numerical or phenotypic differences between floxed and WT cells (data not shown). Similar observations were made when resting splenic NK cells from floxed and WT mice were cotransferred into lymphopenic Rag2−/− × Il2rg−/− mice, followed by tamoxifen treatment (data not shown). Altogether, these data are consistent with the previously ascribed role of Eomes and T-bet in repressing and promoting TRAIL expression, respectively, and in the maintenance of a mature phenotype (5).
Eomes and T-bet are required for the clonal expansion of virus-specific NK cells
To investigate the effects of T-box transcription factors on the various stages of the NK cell response against viral infection, we first induced deletion of Eomes or T-bet immediately before infection to test for their role in clonal expansion of virus-specific NK cells. We isolated splenic Ly49H+ NK cells from Eomesf/f × UbCre-ERT2 or Tbx21f/f × UbCre-ERT2 mice (CD45.2) and cotransferred them with an equal number of WT Ly49H+ NK cells (CD45.1) into Ly49h−/− hosts that were treated with tamoxifen (or oil as a control). Host mice were then infected with MCMV 4 d after adoptive transfer. We found Eomes to be critical for the expansion of Ly49H+ NK cells during MCMV infection, as Eomes−/− NK cells exhibited a marked impairment compared with WT NK cells (Fig. 2A, Supplemental Fig 2). No significant difference in expansion between WT and Eomes−/− NK cells was found in the control group receiving oil alone (Supplemental Fig 2). Activated NK cells deficient in Eomes showed a significant decrease in KLRG1 expression (Fig. 2B) at day 7 postinfection (PI), but similar levels of FLICA staining (data not shown), which detects cells undergoing apoptosis. Although deletion of Eomes did not affect TRAIL expression in splenic or liver NK cells at day 7 PI, a decrease in the CD27+ CD11b+ NK cell population was observed (Fig. 2C).
Similar to Eomes deletion, when T-bet was inducibly ablated following transfer, effector and memory Ly49H+ NK cell numbers were severely diminished compared with WT controls (Fig. 2D, Supplemental Fig. 2). Unlike T-bet deletion in mature NK cells during basal homeostasis (Fig. 1), virus-specific NK cells lacking T-bet exhibited an increase in the percentage of TRAIL+ NK cells and a less mature phenotype (CD27+ CD11b+ KLRG1lo) at day 7 PI (Fig. 2E, 2F), highlighting a role for T-bet in the differentiation and terminal maturation of antiviral effector NK cells. An increase in CD27 expression may be due to a loss in gene repression by T-bet, and not a reversion to a phenotypically distinct immature population (13). A similar phenotype was observed at day 28 PI (data not shown). When deletion of Eomes or T-bet was initiated at day 3 PI instead of at day −4 PI, similar results were observed (data not shown). Altogether, these findings uncover a novel and stage-specific role for T-bet in differentiating NK cells, where T-bet promotes TRAIL expression in resting NK cells but represses its expression in activated NK cells.
To determine whether the decreased expansion of T-bet−/− or Eomes−/− NK cells during viral infection was due to diminished proliferation, we adoptively cotransferred equal numbers of cell trace violet–labeled splenic NK cells from WT and Eomesf/f × UbCre-ERT2 or Tbx21f/f × UbCre-ERT2 mice into Ly49h−/− recipients that were immediately treated with tamoxifen (or oil as a control). Compared to their WT counterparts, a greater percentage of NK cells lacking individual T-box transcription factors had not divided in response to MCMV infection (Fig. 2G, 2H), suggesting that T-bet and Eomes activity in mature NK cells is necessary for the execution of a robust proliferative program. Altogether, these data highlight a nonredundant role for T-box transcription factors during mature NK cell priming for the vigorous proliferation and proper differentiation of virus-specific NK cells.
IL-12 and STAT4 upregulate T-bet in NK cells during MCMV infection
To gain insight into when T-box transcription factors may be exerting influences on the proliferation of virus-specific NK cells, the amount of T-bet and Eomes in Ly49H+ NK cells were measured at different time points during MCMV infection. We found that T-bet expression is upregulated in NK cells at day 2 PI and remains high at effector (day 7 PI) and memory (day 42 PI) time points, whereas Eomes expression is largely unchanged at day 2 PI and downregulated at day 7 and 42 PI (Fig. 3A). The rapid modulation of T-bet levels immediately following MCMV infection suggests a role for proinflammatory cytokine signals in its upregulation. Because IL-12 has been described to upregulate T-bet expression in CD8+ T cells (14, 15), we evaluated the expression of T-bet in NK cells from WT:Il12rb2−/− bone marrow chimeric mice infected with MCMV. In contrast to WT NK cells, we found that T-bet levels were not upregulated at day 2 and 7 PI in Il12rb2−/− NK cells (Fig. 3B). Eomes expression in Il12rb2−/− NK cells did not differ from WT NK cells at various time points during MCMV infection (Supplemental Fig. 2C).
Given that IL-12 induces T-bet, we predicted that the STAT4, which acts downstream of the IL-12 receptor, may directly bind to the gene loci encoding T-bet (Tbx21) in activated NK cells. Analysis of the Tbx21 promoter and enhancer regions revealed several putative STAT4 binding sites (Supplemental Fig. 2D), one of which (site 2) is embedded within a conserved noncoding site. We performed STAT4 ChIP on resting and IL-12 + IL-18–activated NK cells, and found substantial enrichment of STAT4 at site 2 (Fig. 3C, 3D), an enhancer ∼11 Kb upstream of the Tbx21 transcriptional start site. In addition, we demonstrate an increase in the “permissive” histone modification H3K4me3 at the promoter region in activated WT but not Stat4−/− NK cells, which correlates with an increase in Tbx21 transcript levels in the WT but not Stat4−/− NK cells at day 2 PI (Fig. 3D, Supplemental Fig. 2E). Thus, together these data support the direct regulation of T-bet expression in NK cells by IL-12 signaling and STAT4 activity during MCMV infection.
T-bet, but not Eomes, is required for the maintenance of virus-specific memory NK cells
Lastly, we investigated the role of Eomes and T-bet on the maintenance of memory NK cells following MCMV infection. We isolated NK cells from the spleen of Eomesf/f × UbCre-ERT2 or Tbx21f/f × UbCre-ERT2 mice (CD45.2) and cotransferred them with equal numbers of WT NK cells (CD45.1) into Ly49h−/− recipients that were immediately infected with MCMV. At day 19 PI, when memory NK cell numbers were comparable between the two groups, we administered tamoxifen to induce gene deletion in the Eomesf/f × UbCre-ERT2 NK cells. We found that the ratio of WT to Eomes-deleted NK cells did not change for several weeks after tamoxifen administration (Fig. 4A). Furthermore, no changes were observed in the frequency of TRAIL-expressing or CD27+ CD11b+ NK cells (Fig. 4B), and unlike effector NK cells, memory NK cells did not suffer a decrease in KLRG1 expression (Fig. 4C). This lack of a memory maintenance defect in Eomes−/− NK cells is consistent with the observation that Eomes expression was found to be downregulated in virus-specific memory NK cells (Fig. 3A).
In contrast to Eomes ablation, induced deletion of T-bet in memory NK cells led to a significant 3–4-fold decrease in the ratio of T-bet−/− to WT cells in mice receiving tamoxifen compared with controls (Fig. 4D), suggesting a role for T-bet in the persistence of virus-specific memory NK cells. Although only slight changes were observed in the frequency of TRAIL-expressing or CD27+ CD11b+ NK cells (Fig. 4E, Supplemental Fig. 2F, 2G), a significant decrease in the expression of KLRG1 was observed (Fig. 4F, Supplemental Fig. 2G). Previous reports have suggested T-bet to be an important factor in the terminal maturation of NK cells (4), and our data suggests that T-bet is continuously required for the maintenance of a mature NK cell phenotype. Altogether, these data uncover a unique role for T-bet, but not Eomes, in the maintenance of number and phenotype of virus-specific memory NK cells, further highlighting the divergent function of these T-box family transcription factors at different stages of the antiviral response.
Although Eomes and T-bet may play a complementary role in the induction of a proliferative program in NK cells responding to viral infection, our data suggest that only T-bet acts to maintain Ag-experienced NK cells in a mature state. We propose that in addition to TRAIL marking immature NK cells (5, 16) and possibly a distinct NK cell lineage in the liver (6), it may also serve to track NK cell differentiation, as loss of TRAIL was accompanied with increased KLRG1 expression. Interestingly T-bet, but not Eomes, was found to be critical in the maintenance of memory NK cell numbers and phenotype. In contrast to the continuous importance of individual T-box transcription factors throughout the lifespan of NK cells, Nfil3 is a transcription factor required for NK cell development, but dispensable in mature NK cell homeostasis or response to infection (17). How T-bet and Eomes are driving the clonal proliferation of NK cells via a nonredundant mechanism remains to be elucidated. The underlying mechanisms by which T-bet promotes survival of memory NK cells, possibly by regulating IL-15 responsiveness (7), also require further investigation. Nonetheless, these findings highlight a stage-specific influence of individual T-box transcription factors in NK cells responding to MCMV infection.
Our current findings reveal distinct molecular events that influence NK cell responses against viral infection, including the maintenance of a memory NK cell pool, which may inform clinical approaches that couple NK cell-mediated immunity to the treatment of human diseases.
This work was supported by grants from the Medical Scientist Training Program (T32GM07739), the National Institutes of Health (T32AI007621), and the Cancer Research Institute (to S.M.), by a T32 award (CA009149) from the National Institutes of Health (to C.M.L.), by National Institutes of Health Grants T32GM007367 and F30AI113963 (to O.P.), by National Institutes of Health Grants R01AI061699 and R01AI113365 (to S.L.R.), and by the Searle Scholars Program, the Cancer Research Institute, the Burroughs Wellcome Fund, the American Cancer Society, and National Institutes of Health Grants R01AI100874, R01AI130043, and P30CA008748 (to J.C.S.).
The sequences presented in this article have been submitted to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE106137 and GSE106138.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.