Abstract
Ets1-deficient mice develop B and T cells but display a severe defect in the development of the NK cell lineage. In this report, we demonstrate that Ets1 is also required for the development of NK1.1+ T (NK T) cells. We observed significantly decreased numbers of NK T cells in the thymus, spleen, and liver of Ets1-deficient mice. These organs also contained markedly decreased levels of the canonical Vα14-Jα281 TCRα transcript seen in NK T cells. Unlike wild-type NK T cells, Ets1-deficient thymocytes failed to produce detectable levels of IL-4 following anti-CD3 stimulation. The absence of NK T cells in the Ets1-deficient mice was not associated with defective expression of CD1, an MHC class I molecule required for NK T cell development. We conclude that Ets1 defines a novel transcriptional regulatory pathway that is required for the development of both the NK and NK T cell lineages.
Natural killer T cells are a discreet population of T lymphocytes defined by a unique cell-surface phenotype, a restricted TCR repertoire, and specific functional properties following activation through the TCR. These cells typically express TCR β-chains of the Vβ2, Vβ7, and Vβ8 gene families (1, 2, 3) in conjunction with an invariant TCR α-chain composed of Vα14 and Jα281 gene segments (4, 5, 6, 7). NK T cells display a CD44high Mel-14low HSAlow 3G11low cell surface phenotype that is similar to that of an activated effector T cell (1, 2, 3, 8). In addition, like NK cells, NK T cells express membrane-associated receptors such as NK1.1 and IL-2Rβ (1, 9). Just as NK T cells have both T and NK cell cell-surface attributes, they also manifest both T and NK cell effector functions. Following TCR-mediated activation, NK T cells can secrete large amounts of IL-4 and IFN-γ, a characteristic of differentiated Th cells (2, 8, 10). However, they have also been shown to display cytotoxic activity, the mechanism of which is more comparable to that of NK cells (11). Previous studies have suggested that NK T cells have unique and important immunomodulatory roles in vivo, including 1) participating in the elimination of mycobacterial pathogens (12), 2) contributing to tumor rejection (11), 3) providing help to B cells in mediating Ab responses to protozoan parasites (13), and 4) regulating autoimmune responses (14, 15, 16).
Little is currently known about the transcriptional pathways that regulate the development and function of the NK T cell lineage. In this report, we have examined the role of the Ets1 transcription factor in NK T cell development. Ets1 is a member of the Ets family of winged helix-turn-helix transcription factors. Ets proteins bind to conserved purine-rich sequences surrounding a GGA core sequence motif and are important regulators of both vertebrate and invertebrate development (17). Several Ets family members are expressed in lymphoid cells, and many lymphoid-restricted genes contain functionally important Ets binding sites (17).
The generation of Ets1-deficient animals has provided an important reagent for understanding the role of Ets1 in the regulation of lymphoid cell development and function (18, 19, 20). Ets1 is not required for the development of mature T and B cells. However, Ets1-deficient mice display a severe defect in NK cell development and function. Moreover, these mice contain decreased numbers of peripheral CD8+ T cells and increased numbers of IgM-secreting plasma cells. Both CD4+ and CD8+ T cells from the Ets1-deficient animals display a severe defect in TCR-mediated activation that can be rescued by stimulation with phorbol ester plus ionomycin. In this paper we show that Ets1 is also required for the differentiation of the NK T cell lineage and, as such, defines a novel transcriptional pathway that regulates the development of both NK and NK T cells in mice.
Materials and Methods
Mice
Ets1-deficient mice were generated as described previously (20). CD1-deficient mice were generated as previously described and had been backcrossed onto the BALB/c background for seven generations (21).
Antibodies
PE-conjugated anti-NK1.1 (PK136), FITC-conjugated anti-CD8α (53-6.7), and CyChrome-conjugated anti-CD4 (RM4-5), and anti-TCRβ (H57-597) were purchased from PharMingen (San Diego, CA). The hamster anti-mouse CD1 mAb has been described previously (27). The 2.4G2 anti-FcR mAb (22) producing hybridoma and bioreactor supernatant containing hamster anti-mouse CD3ε were kindly provided by Dr. Jeffrey A. Bluestone (University of Chicago, Chicago, IL).
Isolation of lymphocyte populations
Thymus, lymph node, hepatocyte, and splenocyte suspensions were prepared as described previously (19, 20, 21).
Flow cytometric analysis
A total of 106 cells per staining reaction were washed in FACS buffer (0.1% BSA in PBS containing 0.01% sodium azide). Fc receptors were blocked with the addition of 2.4G2 culture supernatant. Cells were stained for 30 min at 4°C and washed in FACS buffer before analyzing on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
RT-PCR analysis of Vα14-Jα281 expression
Five micrograms of total RNA was used to synthesize cDNA by reverse transcription with random hexamer primers. The resulting cDNAs were subjected to 38 cycles of amplification by the PCR (94°C for 1 min, 60°C for 1 min, and 72°C for 2 min) using synthetic oligonucleotide primers specific for either Vα14-Jα281, Vα8-Jα2, or Cα1-Cα2 (21). The amount of template cDNA used in each reaction was normalized to the amount of HPRT mRNA (21). The levels of TCRα cDNA in the samples were normalized to the Cα mRNA amplified with Cα1 and Cα2 primers (21).
In vitro thymocyte stimulation
Triplicate aliquots of 106 thymocytes were stimulated with plate-bound anti-CD3 mAb (10 μg/ml) for 48 h in flat-bottom 96-well plates. Levels of IL-4 in culture supernatants were determined by ELISA using a commercially available kit according to the manufacturer’s instructions (Endogen, Woburn, MA).
Results
Ets1-deficient mice lack NK1.1+ CD4+ T cells
The Ets1-deficient mice were generated on a C57BL/6 × 129/J mixed background. The 129/J strain is of the NK1.2 allele. To identify Ets1 mutant and wild-type mice that carried the NK1.1 allele, peripheral blood from both wild-type and Ets1-deficient animals was analyzed by flow cytometry for NK1.1 expression. Using this protocol, we could detect NK1.1+ cells in the peripheral blood of both the wild-type (3.8% of PBL) and Ets1-deficient (0.5% of PBL) mice (data not shown). All experiments were performed using animals that were determined to express the NK1.1 allele by this flow cytometric assay.
To determine whether NK T cells develop in the Ets1-deficient animals, we analyzed lymphocyte populations from the thymus, spleen, and mesenteric lymph nodes. In addition, lymphocytes were isolated from the liver, a compartment known to be rich in NK T cells in the mouse (23). As shown in Fig. 1⇓A, there were significantly decreased numbers of NK1.1+ CD4+ T cells in both the thymi and spleens of the Ets1-deficient mice. In the liver, as in other lymphoid organs, the majority of the NK T cells express only intermediate levels of the TCR complex. Therefore, we gated on this population of TCR-intermediate lymphocytes (Fig. 1⇓B, top left and right) before analyzing the cells for NK1.1+ expression (Fig. 1⇓B, bottom left and right). NK1.1+ T cells were almost undetectable in the livers of the Ets1-deficient mice. Consistent with this marked decrease in NK1.1+ T cells in the livers of these animals, there was also a significant decrease in the numbers of cells expressing an intermediate level of the TCR β-chain that is characteristic of hepatic NK T cells (Fig. 1⇓B, top left and right). In addition to the decrease in NK1.1+ hepatic T cells, we also observed a significant reduction in the number of TCRintCD4+NK1.1− hepatic lymphocytes in the Ets1-deficient mice. The function of these cells and their relationship to classical NK T cells remains unclear (24). Taken together, these data suggested a severe defect in NK T cell development in the Ets1-deficient mice.
Flow cytometric analysis of NK T cells from wild-type and Ets1-deficient mice. A, Wild type (+/+) and Ets1-deficient (−/−) thymocytes and splenocytes were examined by three color staining with anti-TCR-β-FITC, anti-NK1.1-PE, and anti-CD4-CyChrome. NK1.1+ CD4+ cells are indicated as a percentage of total thymocytes. B, Lymphocytes from perfused livers were subjected to three-color staining as described in A. The top panels are histograms representing TCRβ expression. Cells expressing intermediate levels of TCRβ were gated, and the percentage of this population is shown. The bottom panels show the percentage of NK1.1+ CD4+ T cells in the population of TCRβint cells. Results are representative from at least three mice in each group.
Reduced expression of Vα14-Jα281 transcripts in Ets1-deficient animals
As described above, NK T cells express a limited repertoire of TCR α- and β-chains. The majority of NK T cells express a TCR α-chain encoded by the Vα14-Jα281 gene segments (4, 5). In mice deficient for the Jα281 gene, no NK T cells were observed either in central or peripheral lymphoid organs (11). Similarly, mice deficient for the nonclassical MHC class I molecule, CD1, lack NK T cells and display a marked reduction in Vα14-Jα281 transcripts in the thymus and liver (21, 25). Accordingly, we examined the expression of Vα14-Jα281 transcripts in the livers of Ets1-deficient mice (Fig. 2⇓). There was a significant decrease in the levels of Vα14-Jα281 transcripts in Ets1-deficient livers as compared with wild-type control livers. This decrease was comparable to that observed in livers from the CD1-deficient mice (Fig. 2⇓). Moreover, this decrease was specific for the NK T-associated transcripts, because the wild-type, Ets1-deficient, and CD1-deficient livers contained equivalent levels of Vα8-containing TCRα transcripts that are not associated with NK T cell populations (Fig. 2⇓). Decreased levels of Vα14-Jα281 transcripts were also observed in the thymi and lymph nodes of the Ets1-deficient animals (data not shown). These results were consistent with the NK1.1 flow cytometric data demonstrating a significant decrease in NK T cell numbers in the Ets1-deficient mice.
Semiquantitative PCR analysis of Vα14-Jα281 transcripts in the livers of wild-type, CD1-deficient, and Ets1-deficient mice. Input cDNAs were equalized by the PCR using serial dilutions from each sample and primers to detect HPRT expression. Normalized cDNA samples were then used as templates for the amplification of Cα, Vα8-Cα, and Vα14-Jα281.
Absence of IL-4 production by thymocytes from Ets1-deficient mice
Naive peripheral T cells do not produce IL-4 upon stimulation through the TCR. In contrast, thymic NK T cells have been shown to produce significant quantities of IL-4 following TCR cross-linking (2, 8, 10, 21). To determine whether such IL-4-producing NK T cells were present in the thymi of Ets1-deficient animals, thymocytes were isolated from wild-type and Ets1-deficient mice and cultured on anti-CD3 coated plates for 48 h. Thymocytes from control wild-type animals produced significant quantities of IL-4 following TCR cross-linking in vitro (Fig. 3⇓). In contrast, no IL-4 was detectable in the supernatants from anti-CD3-stimulated Ets1-deficient thymocytes (Fig. 3⇓), suggesting that the NK T cells were absent or nonfunctional in the Ets1-deficient mice.
IL-4 production by Ets1-deficient thymocytes following in vitro stimulation with anti-CD3 mAb. Wild-type (+/+) or Ets1-deficient (−/−) thymocytes were incubated in medium alone or were stimulated with plate-bound anti-CD3 mAb (10 μg/ml). After 48 h of stimulation, supernatants were analyzed for IL-4 by ELISA. Each bar represents the data from an individual mouse. Error bars represent the SD of triplicate wells.
Impaired NK T cell development in Ets1-deficient mice is not due to decreased expression of CD1
The absence of NK T cells in the Ets1-deficient animals suggested that these mice either lack the extrinsic signals required for the development of this lymphocyte population, or are intrinsically incapable of responding to these developmental signals due to the absence of an NK T cell developmental pathway that is transcriptionally regulated by Ets1. It has been demonstrated previously that the nonclassical MHC class I molecule, CD1, can be recognized by hybridomas generated from murine NK1.1+ T cells (26) and that CD1 is required for the development of this lineage in mice (21, 25).
To determine whether the defect in NK T cell development in the Ets1-deficient mice was the result of decreased CD1 expression, thymocytes and splenic B cells from wild-type and Ets1-deficient animals were analyzed for CD1 expression by flow cytometry. There was no significant difference in the levels of cell surface CD1 expression on wild-type and Ets1-deficient lymphocytes (Fig. 4⇓). Ets1-deficient lymphocytes also expressed normal levels of class I MHC as assessed by flow cytometry (data not shown). Thus, the defect in NK T cell development seen in the Ets1-deficient mice is not due to decreased levels of expression of CD1 in these animals.
Flow cytometric analysis of CD1 expression on Ets1-deficient lymphocytes. Thymocytes and splenic B cells from wild-type (WT) or Ets1-deficient (ETS1−/−) mice were analyzed for expression of the nonclassical MHC class I molecule, CD1, by flow cytometry. CD1 staining is represented by the thick black line, and staining with an isotype-matched control (Control Ig) is shown by the thin black line.
Discussion
In the studies described in this report, we have demonstrated that the Ets1 transcription factor is required for the development and/or survival of NK T cells in mice. Previous studies have demonstrated that Ets1 is not required for the development of mature single positive T cells or IgM+ B220+ B cells, but is necessary for the development of NK cells in mice (18, 19, 20). Thus, Ets1 appears to define a transcriptional pathway that distinguishes NK T cells from conventional T and B cell populations. Moreover, these results suggest a close developmental linkage of the NK and NK T cell lineages.
It is not possible from our data to conclude if the defect in NK T cell development observed in the Ets1-deficient mice is cell autonomous or instead reflects a defective Ets1-regulated developmental signal that is required for NK T cell differentiation or survival. We have shown previously that Ets1 is expressed at high levels in wild-type NK cells and T cells (20). However, because it is difficult to purify large numbers of NK T cells away from NK cells, we are currently unable to determine whether Ets1 is also expressed in the NK T cell lineage.
The markedly reduced numbers of NK T cells seen in the thymus, spleen, and liver of the Ets1-deficient mice strongly suggest that the NK T cell defect observed in these animals reflects the deficient production or survival of these cells. We and others have shown previously that Ets1-deficient T cells display a defect in their ability to respond to stimulation through the CD3 components of the TCR complex (19, 20). Although this defect is not sufficient to block the development of mature single positive T cells, more recent studies have demonstrated partial defects in both positive and negative selection of Ets1-deficient TCR transgenic T cells (T. Walunas and J. Leiden, manuscript in preparation). These findings are consistent with a model in which decreased TCR-mediated signaling in the absence of Ets1 partially, but incompletely, blocks T cell maturation and selection. The affinity of the NK T cell TCR for selecting elements such as CD1 remains unknown. However, if this is a low affinity interaction, this same Ets1-related defect in TCR signaling might be predicted to have a more profound effect on NK T cell selection and maturation resulting in the marked reductions in NK T cell numbers seen in the Ets1-deficient animals.
The decreased numbers of NK T cells in the livers and lymph nodes of the Ets1-deficient animals might also reflect defects in the survival or population of peripheral lymphoid organs by NK T cells. Such defects could result from deficient expression of a number of different growth factors, growth factor receptors, or adhesion molecules. Our previous studies have not demonstrated defects in the production of IL-2, IL-15, IL-12, or their receptors in the Ets1-deficient mice (20). Ongoing studies designed to elucidate the normal targets of Ets1 in both lymphocytes and other cell lineages that are important for NK and NK T cell differentiation and survival should help to elucidate the components of the Ets1-dependent pathways(s) that regulate the development and survival of these two important lymphoid cell lineages.
Acknowledgments
We thank R. Doherty for help with the preparation of the manuscript and T. Lis for assistance with the preparation of illustrations.
Footnotes
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↵1 T.L.W. was supported by an institutional National Research Service Award grant from the National Heart, Lung, and Blood Institute (HL-07381-18). This work was supported in part by a grant from the National Institute of Allergy and Infectious Diseases to J.M.L. (AI29673) and from the National Institutes of Health to C.-R.W. (A143407).
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↵2 Address correspondence and reprint requests to Jeffrey M. Leiden, Harvard School of Public Health, Laboratory of Cardiovascular Biology, Building II, Room 117, 677 Huntington Avenue, Boston, MA 02115. E-mail address: leiden{at}cvlab.harvard.edu
- Received December 6, 1999.
- Accepted January 11, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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