IFN-γ–Producing and IL-17–Producing γδ T Cells Differentiate at Distinct Developmental Stages in Murine Fetal Thymus

Kensuke Shibata, Hisakata Yamada, Masataka Nakamura, Shinya Hatano, Yoshinori Katsuragi, Ryo Kominami and Yasunobu Yoshikai
J Immunol March 1, 2014, 192 (5) 2210-2218; DOI: https://doi.org/10.4049/jimmunol.1302145

Abstract

γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ–producing and IL-17–producing γδ T cells developed from DN2 cells, only IFN-γ–producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17–producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17–producing γδ T cells, although a unique subset of IFN-γ–producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.

Introduction

T cells bearing γδTCR are naturally occurring effector T lymphocytes generated in fetal thymus (1, 2). Thymus generates two T cell subsets, expressing αβTCR or γδTCR, both of which are derived from common precursors (3, 4). In mice, hematopoietic stem cells in the fetal liver migrate into the fetal thymus, where interaction between Notch1 and one of the Notch ligands, delta-like 4(Dll4), induces the T cell fate decision of precursors known as CD4−CD8− double-negative (DN) cells (5–7). The DN cells are further classified into four subsets identified by surface expression of CD117, CD44, and CD25: DN1, CD117+CD44+CD25−; DN2, CD117+CD44+CD25+; DN3, CD117−CD44−CD25+; DN4, CD117−CD44−CD25−. During maturation from DN1 to DN4 cells in a stepwise manner, a zinc finger transcription factor, B cell leukemia/lymphoma 11b (Bcl11b), is essential for transition from the early to late DN2 stage (8–10). γδTCR+ cells are detected at the DN2 and DN3 stages in thymus (11), whereas populations that enter the DN4 stage become CD4+CD8+ double-positive (DP) cells and subsequently generate αβ T cells. Given that Bcl11b deficiency abrogates the development of αβ T cells but not γδ T cells, Bcl11b has been thought to be dispensable for the development of γδ T cells (12). In murine thymus, conventional αβ T cells develop as naive cells. Alternatively, murine γδ T cells acquire effector functions to produce IFN-γ, IL-4, and IL-17 in the thymus (1, 2, 13, 14), although the mechanism remains unknown.

Because IFN-γ–producing and IL-17–producing γδ T cells are involved in autoimmune disorders, host defense, wound healing, and tumor surveillance, much attention has been paid to the mechanism of intrathymic functional programming of the two γδ T cell subsets (15). After interacting with thymic epithelial cells, murine γδ T cells acquire functions to produce both IFN-γ and IL-17 within fetal thymus (2, 16, 17). In the thymic microenvironment, it has been reported thus far that cell surface molecules such as γδTCR, Notch, TGF-β receptor, CD27 (receptor for CD70), lymphotoxin β receptor, and skint-1, as well as intracellular molecules such as inhibitor of DNA binding (Id)3, Sry-related HMG box 13, hairy and enhancer of split homolog-1, B lymphoid kinase, and promyelocytic leukemia zinc finger (PLZF), are involved in intrathymic functional differentiation of IFN-γ–producing and IL-17–producing γδ T cells (2, 14, 16–23). However, it remains to be determined at which stage the functional programming of γδ T cells occurs during T cell development.

In the present study, we compared the functions of γδ T cells derived from CD117high DN2 and CD117− DN3 cells in murine fetal thymus by in vitro T cell differentiation system for IFN-γ–producing and IL-17–producing γδ T cells, which we and another group previously used (16, 20). We demonstrated that CD117high DN2 cells but not CD117− DN3 cells had a function to generate IL-17–producing γδ T cells, whereas IFN-γ–producing γδ T cells were generated from both precursors. Single-cell analysis revealed that CD117high DN2 cells were heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Precursors of IL-17–producing γδ T cells were highly enriched in CD117high DN2 cells. Inactivation of Bcl11b selectively abrogated the development of IL-17–producing γδ T cells, but not a unique subset of IFN-γ–producing γδ T cells with high PLZF expression. Thus, we reveal that IFN-γ–producing and IL-17–producing γδ T cells are generated at different developmental stages in fetal thymus.

Materials and Methods

Mice

Pregnant C57BL/6 (B6) mice were purchased from SLC (Shizuoka, Japan). Cβ-deficient mice were purchased from The Jackson Laboratory. Transgenic γ-chain mice were generated by injection of functionally rearranged Vγ6Jγ1Cγ1, which originated from a hybridoma clone (16) with the IRES-EGFP cassette derived from the pIRES2-EGFP vector (Clontech). The transgene was driven by a chicken β-actin promoter from the pCAGGS vector (provided by J. Miyazaki, Osaka University, Osaka, Japan). Cγ-deficient mice were provided by M. Nanno (Yakult Central Institute for Microbiological Research, Tokyo, Japan). Bcl11b-deficient and Bcl11bflox/flox mice were provided by R. Kominami (Niigata University, Niigata, Japan) (12, 24). Rag-1-Cre mice were provided by T. Rabbitts (Leeds Institute of Molecular Medicine, Leeds, United Kingdom) courtesy of K. Akashi (Kyushu University, Kyushu, Japan). This study was approved by the Committee of Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University. Experiments were carried out under the control of the Guidelines for Animal Experiments.

Abs and flow cytometric analysis

PE-conjugated anti–mIFN-γ (XMG1.2) and anti-Vδ6.3/2 (8F4H7B7) mAbs, Alexa 647–conjugated anti–IL-17 (TC11-18H10) mAb, and allophycocyanin-conjugated anti-CD8α (53-6.7) and anti-CD25 (PC61) mAbs were purchased from BD Biosciences. FITC-conjugated anti-CD45.2 (104) and anti-Sca1 (D7) anti–MHC class II (M5/114.15.2) mAbs, PE-conjugated anti-CD4 (GK1.5), anti-CD117 (2B8), and anti-CD122 (TM-b1) mAbs, PerCP-eFluor 710–conjugated anti-TCRδ (GL3) mAb, allophycocyanin-conjugated anti–IFN-γ (XMG1.2) mAb and eFluor 660–conjugated anti–T-bet (eBio4B10) mAbs were purchased from eBioscience (San Diego, CA). FITC-conjugated anti-Vγ1 (2.11) mAb, PE-conjugated anti-CCR6 (29-2L17), anti-TCRδ (GL3), and anti-NK1.1 (PK136) mAbs, allophycocyanin-conjugated anti-Vγ4 (UC3-10A6) and anti-CD24 (M1/69) mAbs, PE/Cy7-conjugated anti–IL-4 (11B11) mAb, and allophycocyanin/Cy7-conjugated anti-CD45 (30-F11) mAb were purchased from BioLegend. Single-cell suspensions were prepared from various tissues as described previously (1). Stained cells were analyzed on a FACSCalibur or FACSVerse flow cytometer (BD Biosciences). We added propidium iodide (1 μg/ml) to the cell suspension just before running a flow cytometer to detect and exclude dead cells for an analysis of surface staining. The data were analyzed using FlowJo software version 9.6.2 (Tree Star). For detecting cytokines intracellularly, cells were stimulated with 25 ng/ml PMA (P-8139; Sigma-Aldrich) and 1 μg/ml ionomycin (I-0634; Sigma-Aldrich) for 4 h at 37°C. For the last 3 h of incubation, 10 μg/ml brefeldin A (B-7651; Sigma-Aldrich) was added. After cells were stained with various mAbs for 20 min at 4°C, intracellular staining was performed according to the manufacturer’s instruction (BD Biosciences). We used Heilig and Tonegawa’s (25) nomenclature for TCRγ genes.

ELISA

After single-cell suspensions were prepared from fetal liver or fetal thymus (embryonic day [E]17), lineage+ cells were stained using lineage cell detection mixture (biotinylated Abs of CD5 [53-7.3], B220 [RA3-6B2], CD11b [M1/70], Gr-1 [RB6-8C5], 7-4, Ter-119, CD3ε [145-2C11], DX5) and depleted by MACS column according to the manufacturer’s instructions (Miltenyi Biotec). Lineage−Sca1+c-Kit+ cells from the fetal liver and DN2 (CD117+CD25+), DN3 (CD117−CD25+), DP (CD4+CD8α+), and γδ T (TCRδ+) cells from the fetal thymus were sorted by FACSAria (BD Biosciences). The sorted cells were stimulated with PMA and ionomycin for 24 h in a CO2 incubator at 37°C. After stimulation, IL-17 and IFN-γ in the supernatant were analyzed by the DuoSet ELISA Development System (R&D Systems) according to the manufacturer’s instructions.

Coculture of stromal cells

To induce T cell differentiation in vitro, TSt-4 thymic stromal cells expressing murine Dll4 gene (TSt-4/Dll4) were used as described previously (8). Lineage− DN cells from fetal thymocytes (E17) were cocultured on a monolayer of TSt-4/no or TSt-4/Dll4 cells in 24-well plates for the indicated days. The culture was performed without additional cytokines, and half of the medium was changed every 5 d.

Gene expression analysis

Total RNA from sorted cells was extracted by the RNeasy Micro kit (Qiagen). Efficacy of cell-sorting was constantly >98%. The first-stranded cDNA synthesis was done using SuperScript Ι (Invitrogen) according to the manufacturer’s instructions.

Gene-specific primers were used as follows (forward, reverse): Id3, 5′-ACTCAGCTTAGCCAGGTGGA-3′, 5′-TCAGTGGCAAAAGCTCCTCT-3′; PLZF, 5′-CCCAGTTCTCAAAGGAGGATG-3′, 5′-TTCCCACACAGCAGACAGAAG-3′; Id2, 5′-CTGGACTCGCATCCCACTAT-3′, 5′-CTCCTGGTGAAATGGCTGAT-3′; GAPDH, 5′-GGCAAATTCAACGGCACA-3′, 5′-GTTAGTGGGGTCTCGCTCTG-3′. Quantitative RT-PCR was performed on an ABI Prism thermal cycler (Applied Biosystems) using SYBR Premix Ex Taq (RP041A; Takara). The 2−ΔΔCt equation was used to calculate the relative expression of target genes against that of GAPDH. In some experiments, the relative abundance of each message was calculated as: 2−(Ct gene − Ct GAPDH), where Ct represents the threshold cycle.

Statistics

Statistical significance was calculated by the Student t test using Prism software (GraphPad Software, San Diego, CA). A p value <0.05 was considered statistically significant.

Results

The IL-17–producing function is predominantly found in γδ T cells but not in T cell precursors in fetus

A recent study showed that γδTCR− populations containing T cell precursors had the IL-17–producing function in fetal thymus (26). However, there was no direct testing of whether the T cell precursors acquired the IL-17–producing and IFN-γ–producing functions in the fetal thymus. We noticed that, in contrast to γδTCR expression, the CD117, CD25, and CD44 expression profiles to identify DN populations were not stable after mitogen stimulation (data not shown). Therefore, to analyze functional properties of the T cell precursors, each population was electronically sorted from fetus (E17) and then stimulated with PMA and ionomycin. After the stimulation, production of IFN-γ and IL-17 by each population was analyzed by ELISA (Fig. 1A). We found that all T cell precursors as well as DP cells were not functionally committed to IL-17 producers. The IL-17–producing function was predominantly found in γδ T cells, although the IFN-γ–producing function had already been detected in DN2 and DN3 cells as well as γδ T cells. The IFN-γ–producing functions were observed in DN2 and DN3 cells of Rag1-deficient mice (E17) (Supplemental Fig. 1B). After depleting lineage+ cells, CD25+ DN2 and DN3 cells from both Rag1-deficient and B6 mice (E17) did not contain NK1.1+ NK cells (Supplemental Fig. 1A, 1C). The DN2 and DN3 cells expressed the transcriptional repressor Id2 (Supplemental Fig. 1D), which is essential for the development of NK1.1− NK cell precursors as well as NK1.1+ NK cells (27–29). The high Id2 expression in DN3 cells was also reported in adult mice (30). Taken together, there might be a possibility that NK cell precursors were present in DN cells, but we did not further analyze the subsets.

FIGURE 1.
FIGURE 1.

IL-17–producing γδ T cells develop directly from DN2 cells but not DN3 cells. (A) CD5, B220, CD11b, Gr-1, Ter119, 7-4, CD3ε, and DX5 were used as lineage markers. Lineage−Sca1+c-Kit+ cells, lineage− DN2 and DN3 cells, DP cells, and γδ T cells from fetal livers or fetal thymi of B6 mice (E17) were sorted by FACSAria. After stimulation of each subset with PMA and ionomycin for 24 h, cytokine production in the supernatants was analyzed by ELISA. The data are presented as means ± SD of cytokine productions of five individual wells and are representative of four independent experiments. (B) The dot plots show lineage− CD117high DN2 and CD117− DN3 cells before sorting. The histogram indicates the expression profile of CD44 in DN2 and DN3 cells. (C–E, G, and H) Sorted 1,000 CD117high DN2 and 10,000 CD117− DN3 cells from fetal thymi of B6 mice (E17) were cocultured on a monolayer of stromal cells in the presence (TSt4/Dll4) of Notch signaling without additional cytokines. (E) For the coculture on a monolayer of stromal cells in the absence (TSt4/no) of Notch signaling, 10,000 DN2 cells were used. (C–E) After the coculture, IFN-γ–producing and IL-17–producing γδ T cells were intracellularly analyzed after stimulation with PMA and ionomycin (C, D) at each time point or (E) on day 14. (C) Bar graphs show means ± SD of the absolute numbers of total (upper), IL-17+ (middle), and IFN-γ+ (low) γδ T cells (n = 4). (D and E) Representative dot plots after gating on CD45.2+γδTCR+ cells are shown. The numbers in the quadrants show the percentages within γδ T cells. (G) The graph shows the mean fluorescence intensity (MFI) ± SD of IL-7Rα expression in γδ T cells (n = 6). (H) After sorting of CD122+ γδ T cells, relative expressions of Id3 and PLZF to GAPDH were quantitatively analyzed by quantitative RT-PCR. The bar graphs show means ± SD of the expressions of Id3 and PLZF (n = 5). (F) Notch1 expression of IFN-γ–producing and IL-17–producing γδ T cells in neonatal thymi of B6 mice (1-d-old) was analyzed after stimulation with PMA and ionomycin. The graph shows the MFI ± SD of Notch1 expression in γδ T cells (n = 9). (B–H) The data are representative of three independent experiments. (A, F, and G) Statistical significance was determined by an unpaired Student t test. **p < 0.01, ***p < 0.001.

IL-17–producing γδ T cells develop directly from DN2 cells but not DN3 cells

γδ T cell development occurs at the DN2 and DN3 stages in thymus (11). We first compared functions of γδ T cells derived from DN2 and DN3 cells (E17) using an in vitro T cell differentiation system (8, 16, 20). After the culture of sorted CD117high DN2 and CD117− DN3 cells on Dll4-expressing thymic stromal cells, the absolute number of γδ T cells gradually increased and reached a peak at 13 d (Fig. 1B, 1C). IL-17–producing γδ T cells were detected mostly in DN2 cell–derived γδ T cells but not in DN3 cell–derived γδ T cells, suggesting the direct development of IL-17–producing γδ T cells from the DN2 stage (Fig. 1B–D). In contrast, IFN-γ producers were detected in γδ T cells derived from both precursors. Nevertheless, different γδ T cell repertoires develop at early and late embryonic stages (31), suggesting the possibility that T cell precursors have different potentials for the development of IL-17–producing and IFN-γ–producing γδ T cells depending on the fetal ages rather than the developmental stages. To test this, we compared the potentials of DN2 and DN3 cells between the early (E15) and late (E17) embryonic stages. We found that, irrespective of the fetal ages, DN3 cells lost the potential to develop IL-17–producing γδ T cells as found in those on E17, whereas DN2 cells were able to generate both IFN-γ–producing and IL-17–producing γδ T cells (Supplemental Fig. 2A, 2B). The potentials in DN2 cells were also observed in DN1 cells from both early and late embryonic stages. These results suggest that the development of IL-17–producing and IFN-γ–producing γδ T cells in fetal thymus is a preprogrammed event at developmental stages of DN cells. We previously reported that Notch signaling was indispensable for the development of IL-17–producing γδ T cells (16). Consistent with the previous finding, in the absence of Notch signaling, IFN-γ–producing γδ T cells but not IL-17–producing γδ T cells developed from CD117high DN2 cells (Fig. 1E). Indeed, significantly higher levels of Notch1 expression were observed in IL-17–producing γδ T cells than in IFN-γ–producing γδ T cells (Fig. 1F). After functional differentiation, IL-17–producing γδ T cells expressed a high level of IL-7Rα (32). IL-7Rα expression was higher in γδ T cells from CD117high DN2 cells than in those from CD117− DN3 cells (Fig. 1G). These results suggest that Notch signaling is indispensable for the development of IL-17–producing γδ T cells directly from DN2 cells, in contrast to the development of IFN-γ–producing γδ T cells from DN3 cells independently of Notch signaling partly via Id3 as described previously (33). Additionally, PLZF was also shown to induce the development of IFN-γ–producing γδ T cells (23). Therefore, we next examined the expression levels of Id3 and PLZF in IFN-γ–producing γδ T cells from different precursors. For this purpose, IFN-γ–producing γδ T cells were identified by surface expression of CD122, which correlated well with the expression of T-bet, a master regulator for the functional differentiation to IFN-γ producers in T cells and NK cells (1, 14, 17, 34) (Supplemental Fig. 2C). We found that the higher expression of Id3 to that of PLZF was clearly observed in CD122+ IFN-γ–producing γδ T cells from DN3 cells but not from DN2 cells (Fig. 1H). These results suggest the different requirement of PLZF and Id3 in the development of IFN-γ–producing γδ T cells between DN2 and DN3 stages.

DN2 cells contain heterogeneous precursors for IL-17–producing γδ T cells

Next, we examined functional properties of individual CD117high DN2 cells to generate IFN-γ–producing or IL-17–producing γδ T cells. In this study, we focused on a fetal thymus-derived Vγ6+ γδ T cell repertoire, which was previously reported as one of the major IL-17–producing γδ T cell subsets (1). We generated mice in which all γδ T cells express the Vγ6 chain (hereafter designated as γ6TgCγKO mice) by crossing mice transgenic (Tg) for rearranged γ6 gene with Cγ knockout (KO) mice. To lower the frequency of αβ T lineage cells, γ6TgCγKO mice were further crossed with CβKO mice (hereafter designated as γ6TgCγKOCβKO mice). As compared with wild-type fetus, CD117high DN2 and CD117− DN3 cells from γ6TgCγKOCβKO fetus maintained similar potentials to develop IL-17–producing and IFN-γ–producing γδ T cells (Supplemental Fig. 3A, 3B). In mice transgenic for the γ-chain, the γ-chain could be paired with pTα to induce DP cells (35). In fact, after the coculture of single DN2 cells on Dll4-expressing thymic stromal cells, progeny cells from 65 individual DN2 cells expressed the TCRδ chain on the cell surface, suggesting bona fide γδ T cells (Supplemental Fig. 3C). A single-cell analysis revealed that the γδTCR-expressing progeny cells were categorized into mainly three types based on their functions: no producers, IFN-γ and IL-17 producers (not both), and only IFN-γ producers (Fig. 2A, 2B). On the one hand, 45% of DN2 cells were functionally differentiated to IFN-γ–producing and IL-17–producing γδ T cells. On the other hand, 32% of DN2 cells were differentiated to only IFN-γ–producing γδ T cells. Taken together, CD117high DN2 cells were heterogeneous populations with different potentials to develop IL-17–producing γδ T cells.

FIGURE 2.
FIGURE 2.

DN2 cells have different potentials for developing IL-17–producing γδ T cells. (A and B) For single-cell analysis, sorted CD117high DN2 cells from fetal thymi of γ6TgCγKOCβKO mice (E17) were plated in a dilution of 0.3 cells per well on a monolayer of TSt4/Dll4 stromal cells and cocultured for 14 d. (A) After the coculture, IFN-γ–producing or IL-17–producing γδ T cells were analyzed after stimulation with PMA and ionomycin. The numbers in the quadrants indicate the percentages of IFN-γ+ or IL-17+ cells within γδ T cells. In this study, cells that produced IFN-γ or IL-17 <1% are considered to be no producers. (B) The pie chart shows the percentage of functionally different γδTCR+ progeny cells from individual DN2 cells. (C–E) After the coculture of sorted 1000 DN2 and 1000 DN2/3 cells from fetal thymi of B6 mice (E17) on TSt4/Dll4 stromal cells, IFN-γ+ and IL-17+ γδ T cells were analyzed after stimulation with PMA and ionomycin. (C) The dot plots show lineage− CD117high DN2 and CD117int DN2/3 cells before sorting. The histogram indicates the expression profile of CD44 in CD117high DN2 and CD117int DN2/3 cells. (D) Representative dot plots are shown after gating on γδ T cells. The numbers in the quadrants indicate the percentages of IFN-γ+ and IL-17+ cells in γδ T cells. (E) The graphs show means ± SD of the percentages of IFN-γ+ and IL-17+ cells in γδ T cells (n = 4). (C–E) The data are representative of three independent experiments. The statistical significance was determined by an unpaired Student t test. ***p < 0.001.

Because CD117 expression decreases along with developmental progression from the DN2 to DN3 stages, DN2 cells contain various expression levels of CD117 (Fig. 2C). To more carefully analyze subpopulations of DN2 cells, electronically sorted CD117int DN2/3 cells were cultured on Dll4-expressing stromal cells. Consistent with a previous study (9), CD117int DN2/3 cells expressed a lower level of CD44 than that in CD117high DN2 cells (Fig. 2C). We found that CD117int DN2/3 cells had less potential than CD117high DN2 cells to generate IL-17–producing γδ T cells (Fig. 2D, 2E). These results suggest that precursors of IL-17–producing γδ T cells are highly enriched in CD117high DN2 cells and gradually decrease with developmental transition from DN2 to DN3 cells.

Bcl11b is a critical factor for the development of IL-17–producing γδ T cells at the DN2 stage

Recently, Bcl11b was identified as an essential molecule for the development of αβ T cells at the DN2 stage, where γδ T cells were able to develop in the absence of Bcl11b (8–10, 12). Although the functions of Bcl11b in γδ T cells were not known, it was reported that IL-17–producing γδ T cells expressed a higher level of the Bcl11b transcript than did IFN-γ–producing γδ T cells (17). That previous finding spurred us to examine functional properties of Bcl11b-deficient γδ T cells. Consistent with previous studies (8, 9), in Bcl11b-deficient mice, αβ T cell development was completely blocked at the DN2 stage before transition to DP cells (Supplemental Fig. 4A, 4B), although γδ T cell development was clearly detected (Fig. 3A, 3B, Supplemental Fig. 4C). Comparative RNA expression analysis revealed that Bcl11b deficiency had opposite effects on expressions of TCRVγ4 and TCRγ5 in DN2 cells (9). Consistently, in the absence of Bcl11b, the proportion of Vγ5+ cells within γδ T cells increased, whereas that of Vγ4+ γδ T cells markedly decreased (Supplemental Fig. 4C). Major IL-17–producing γδ T cell subsets in neonates were mainly cells expressing Vγ4 and Vγ6 (1, 26). The Vγ4+ and Vγ4− γδ T cell subsets expressing CCR6, a marker for IL-17 producers (36), were virtually absent in Bcl11b-deficient mice (Fig. 3A). The complete loss of CCR6+ γδ T cells was not related to the maturation status identified by CD24 expression (Fig. 3A). In agreement with this finding, functional differentiation of γδ T cells to IL-17 producers but not IFN-γ producers was completely blocked in the absence of Bcl11b (Fig. 3B). Therefore, Bcl11b was essential for the development of IL-17–producing γδ T cells at the DN2 stage.

FIGURE 3.
FIGURE 3.

Bcl11b is indispensable for the development of IL-17–producing γδ T cells in neonates. Single-cell suspensions of neonatal thymocytes of Bcl11b+/+, Bcl11b+/−, and Bcl11b−/− mice (1-d-old) were prepared. (A) Representative dot plots after gating on γδTCR+ cells are shown. (B) After gating on γδTCR+ cells (upper), production of IFN-γ and IL-17 (lower) was analyzed after stimulation with PMA and ionomycin. The numbers in the quadrants show the percentages of positive cells within γδ T cells. The data are representative of three independent experiments.

IFN-γ–producing γδ T cells directly from DN2 and DN3 cells highly express PLZF and Id3, respectively

Bcl11b-sufficient γδ T cells have been reported to receive agonistic TCR signals that increase Id3 expression and become IFN-γ producers from DN3 cells (14, 33) (Fig. 1H). In contrast, Bcl11b deficiency failed to increase expression of T cell–associated genes such as CD3γ, CD3ε, lck, and ZAP70 in DN cells (8–10). We found a significant reduction of Id3 expression in Bcl11b-deficient γδ T cells (Fig. 4A), although IFN-γ–producing γδ T cells were able to develop in Bcl11b-deficient mice (Fig. 3B). This finding was supported by a previous study showing that IFN-γ–producing Vγ1+/Vδ6.3+ γδ T cell subsets developed in the absence of Id3 (37). In fact, Vγ1+/Vδ6.3+ γδ T cells acquired the IFN-γ–producing function via PLZF (23). Attenuation of γδTCR-mediated signals enhanced PLZF-expressing Vγ1+/Vδ6.3+ γδ T cells (38). Therefore, we questioned whether Bcl11b deficiency altered the development of Vγ1+/Vδ6.3+ γδ T cells. We found that, in the absence of Bcl11b, Vγ1+/Vδ6.3+ γδ T cells developed concomitantly with increased PLZF expression (Fig. 4A, 4B). This suggested that, in contrast to IL-17–producing γδ T cells, a significant fraction of IFN-γ–producing γδ T cells was able to develop directly from Bcl11b-deficient DN2 cells partly via a PLZF-dependent mechanism. Taken together, functional maturation of IFN-γ–producing γδ T cells occurs directly from both DN2 and DN3 cells but possibly via different functional differentiation pathways in a PLZF- and Id3-dependent manner, respectively (see Fig. 7). Similar to the case of Bcl11b-deficient DN cells (8–10), Bcl11b-deficient γδ T cells increased the Id2 expression (Fig. 4A), suggesting the possibility that Id2 has a role in the development of IFN-γ–producing γδ T cells directly from DN2 cells. We also found that the absolute numbers of Vγ1+Vδ6.3+ γδ T cells as well as total γδ T cells decreased significantly in the absence of Bcl11b (Fig. 4C). Thus, a Bcl11b-mediated signal might be required for the development and/or maintenance of IFN-γ–producing γδ T cells and other γδ T cell populations directly generated from DN2 cells.

FIGURE 4.
FIGURE 4.

IFN-γ–producing γδ T cells expressing a high level of PLZF develop independently of Bcl11b. (A) After sorting of γδ T cells from neonatal thymocytes of Bcl11b+/+ (wild-type [WT]) and Bcl11b−/− (KO) mice (1 d old), relative expressions of Id3, PLZF, and Id2 to GAPDH were quantitatively analyzed by quantitative RT-PCR. The bar graphs show means ± SD of the expressions of Id3, PLZF, and Id2 (n = 3). The statistical significance was determined by an unpaired Student t test. *p < 0.05. (B) Representative dot plots are shown after gating on γδTCR+ cells. (C) The bar graphs show means ± SD of the absolute numbers of γδ T cells (left) and Vγ1/Vδ6.3+ γδ T cells (right) (n = 4). (A–C) The data are representative of three independent experiments. The statistical significance was determined by an unpaired Student t test. *p < 0.05.

The lack of IL-17–producing γδ T cells in the absence of Bcl11b is likely to be cell-intrinsic

The above results suggest that Bcl11b was selectively required for the development of IL-17–producing γδ T cells but not a subset of IFN-γ–producing γδ T cells in fetal thymus. Nevertheless, because germline inactivation of Bcl11b had a severe defect in organization of cortex and medulla in thymus (12), we could not exclude the possibility that the loss of IL-17–producing γδ T cells was due to extrinsic effects. To test this, we generated mice in which Bcl11b was conditionally deleted in γδ T cells by overexpression of Cre recombinase under the control of Rag1 promoter. Similar to the case of neonates, we found complete loss of DP cells in the thymus of conditionally Bcl11b-deficient mice (data not shown). The cell-intrinsic defect of Bcl11b expression significantly reduced the percentage of CCR6+ γδ T cells and selectively blocked the development of IL-17–producing γδ T cells in the thymus as well as in the periphery (Fig. 5). In clear contrast, comparable or slightly increased numbers of IFN-γ–producing γδ T cells with a NK cell lineage marker were present in conditionally Bcl11b-deficient mice as previously described (Figs. 5B, 5C, 6A) (10). In the absence of Bcl11b, PLZF expression was significantly increased in γδ T cells (Fig. 4A). In Vγ1+/Vδ6.3+ γδ T cells as well as NK1.1+ αβ T cells, overexpression of PLZF increased the functions to produce IFN-γ and IL-4 (38, 39). Consistently, the increased PLZF expression due to the Bcl11b deficiency slightly enhanced both IFN-γ–producing and IL-4–producing functions in Vγ1+/Vδ6.3+ γδ T cells (Fig. 6B). These results suggest that cell-intrinsic Bcl11b expression is required for the development of IL-17–producing γδ T cells, but not a DN2-derived NK1.1+ IFN-γ–producing γδ T cell subset, which expressed high Id2 and PLZF. Nevertheless, it remains to be explored whether Bcl11b-independent thymocytes might have roles in the generation of the thymic microenvironment, which supports the development of IFN-γ–producing but not IL-17–producing γδ T cells.

FIGURE 5.
FIGURE 5.

IL-17–producing γδ T cells are absent in the periphery of conditional Bcl11b-deficient mice. Single-cell suspensions were prepared from the thymus, spleen, peritoneal cavities (PEC), and lung from Bcl11bflox/flox (Cre−) or Rag-1-Cre Bcl11bflox/flox (Cre+) mice (3 wk old). (A) The graphs show means ± SD of the percentages of CCR6+ cells within γδ T cells (n = 3). (B and C) IFN-γ+ and IL-17+ γδ T cells were analyzed after stimulation with PMA and ionomycin. (B) Representative dot plots are shown after gating on γδTCR+ cells. The numbers in the quadrants indicate the percentages of cells positive for IFN-γ or IL-17. (C) The bar graphs show means ± SD of the absolute numbers of IFN-γ+ or IL-17+ γδ T cells (n = 3). The data are representative of three independent experiments. (A and C) The statistical significance was determined by an unpaired Student t test. *p < 0.05, **p < 0.01.

FIGURE 6.
FIGURE 6.

Bcl11b-deficient γδ T cells share the features with NK lineage cells. (A and B) Single-cell suspensions of thymocytes and splenocytes from Bcl11bflox/flox (Cre−, black line) or Rag-1-Cre Bcl11bflox/flox (Cre+, filled) mice (4 wk old) were prepared and (A) analyzed after gating on γδ T cells. (B) Splenocytes were analyzed intracellularly after stimulation with PMA and ionomycin (P/I). The representative dot plots are shown after gating on Vγ1+Vδ6.3+ γδ T cells. The numbers in the quadrants show the percentage of IFN-γ+ and/or IL-4+ cells. The data are representative of three independent experiments.

Discussion

In the present study, we demonstrated that IFN-γ–producing and IL-17–producing γδ T cells emerge from different DN stages in murine fetal thymus. In support of our results, microarray analysis revealed that such molecular heterogeneity of effector γδ T cell subsets exists in thymus (40). IL-17–producing γδ T cells develop directly from DN2 cells in a Bcl11b-dependent manner. Thus, Bcl11b expressed in T cell precursors at the DN2 stage is indispensable not only for the development of αβ T cells but also for the development of IL-17–producing γδ T cells during T cell development (8–10, 12). We also found that different types of IFN-γ–producing γδ T cells develop directly from DN2 and DN3 cells, respectively. Taken together, because γδ T cells have been considered to develop through the DN3 stage where γδTCR-mediated signaling regulates the functional cell fate of γδ T cells to IFN-γ or IL-17 producers (15), our finding provides a novel insight into T lymphopoiesis.

γδ T cell development begins at an early embryonic stage with Vγ5+ γδ T cells, followed by other repertoires such as Vγ6+, Vγ4+, Vγ1+, and Vγ7+ cells. By engaging skint-1 on thymic epithelial cells, the Vγ5+ γδ T cells acquire a function to produce IFN-γ (17). However, skint-1 deficiency increases the Bcl11b transcript and changes the function of Vγ5+ γδ T cells into IL-17 producers. This link between the Bcl11b expression profile and IL-17–producing function in Vγ5+ γδ T cells matches our results perfectly. The Bcl11b expression is strongly upregulated from the DN2 stage (41, 42). CD117high DN2 cells are subdivided into two stages based on the Bcl11b expression level (8, 9). The heterogeneous expression of Bcl11b in CD117high DN2 cells may explain the different potentials to generate IL-17–producing γδ T cells. Interestingly, the Bcl11b expression in DN2 cells correlates well with Lck expression, a component of TCR-mediated signaling (Fig. 7) (8). Note that, during αβ T cell development, Bcl11b is required for positive selection by augmenting proximal TCR signaling (43). Hence, it would be interesting to examine whether proximal TCR signaling regulated by Bcl11b may contribute to acquisition of the IL-17–producing function in γδ T cells at the DN2 stage, as is the case for Id3-dependent functional maturation of Ag-specific IFN-γ–producing γδ T cells from the DN3 stage (33). Intriguingly, DN3 cells expressed Bcl11b but lost the potential to develop IL-17–producing γδ T cells (9, 10), suggesting the presence of an as yet unknown suppressive mechanism. Furthermore, a recent study showed that some γδ T cells migrated to the peripheral tissues as naive cells and functionally differentiated to IL-17 producers after Ag encounter (44). It remains unclear whether the development of Ag-induced IL-17–producing γδ T cells from naive cells requires Bcl11b.

FIGURE 7.
FIGURE 7.

Illustration of functional differentiation of γδ T cells in fetal thymus. The top bars show changes of expression patterns of CD117, CD44, CD25, and GFP under the control of Lck promoter (designated as pLck-GFP) (8) along with T cell development in the fetal thymus.

Another notable finding in the present study is that at least two types of IFN-γ–producing γδ T cells develop in fetal thymus in terms of Bcl11b dependency. Because Bcl11b is responsible for transition from the early to late DN2 stage, IFN-γ–producing γδ T cells derived from DN3 cells may be dependent on Bcl11b. The Bcl11b-dependent, DN3-derived, IFN-γ–producing γδ T cells requires the TCR/Id3 axis for their functional maturation (14, 33). In the absence of Bc11b, an appreciable number of IFN-γ–producing γδ T cells develop from the early DN2 stage with significantly increased PLZF expression. PLZF was predominantly expressed by Vγ1+Vδ6.3+ γδ T cells (38). Attenuation of TCR-mediated signals increased both PLZF expression and frequency of IFN-γ–producing cells in Vγ1+Vδ6.3+ γδ T cells (38). Nevertheless, crosslinkings of γδTCR were able to enhance PLZF expression in γδ T cells (23). Genetic alteration of δ-chain slightly impaired the development of IFN-γ–producing PLZF+Vγ1+ γδ T cells (45). Thus, it remains to be resolved whether TCR-mediated signals are involved in the development of Bcl11b-independent, DN2-derived, IFN-γ–producing γδ T cells.

Recently, as a new group of lymphoid populations, innate lymphoid cells (ILCs) have been identified (46). Unlike T and B cells, all ILCs lack rearranged Ag receptors yet produce various types of cytokines including IFN-γ and IL-17, which are secreted immediately after stimulation with IL-12 and IL-23 similar to the features of IFN-γ–producing and IL-17–producing γδ T cell subsets. This raises the possibility that the IFN-γ–producing function observed in TCR− DN cells is due to contamination of IFN-γ–producing ILC1s, which are clearly distinct from NK cells (47).

Since 450 million years ago, TCR α, β, γ, and δ genes have been highly conserved in jawed vertebrates, all of which have a thymus (48). Bcl11b or its homolog was highly expressed in the thymus of jawed vertebrates (49). Moreover, in a primitive jawless vertebrate, sea lamprey, T-like lymphocytes expressing variable lymphocyte receptor A were found (49, 50). Interestingly, variable lymphocyte receptor A+ lymphocytes expressed a high level of Bcl11b transcript and produced IL-17 in response to T cell mitogen, suggesting an evolutionarily conserved correlation between Bcl11b and the development of IL-17–producing lymphocytes. In fact, Bcl11b is essential for the development of IL-17–producing γδ T cells in mice. Moreover, two IFN-γ–producing γδ T cell subsets develop from DN2 and DN3 cells in Bcl11b-independent and Bcl11b-depependent mechanisms, respectively. Hence, our study provides a novel insight into functional differentiation of IL-17–producing and IFN-γ–producing γδ T cells from preprogrammed DN cells possibly in large part as a common feature in T lymphopoiesis as well as αβ T cell development in higher vertebrates (8–10, 12), and it advances the understanding of T cell biology, which can be useful for therapeutic applications.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Mihoko Okubo, Miki Kijima, and Akiko Yano for secretarial assistance.

Footnotes

  • This work was supported by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, which is a project launched and commissioned by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), by a Grant-in-Aid from the Japan Society for Promotion of Science, and by grants from Japanese Ministry of Education, Science and Culture (to Y.Y. and K.S.). K.S. receives support from the Kaibara Morikazu Medical Science Promotion Foundation and the Takeda Science Foundation. This work was supported in part by Grants for Excellent Graduate Schools, MEXT, Japan.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    B6
    C57BL/6
    Bcl11b
    B cell leukemia/lymphoma 11B
    Dll4
    delta-like 4
    DN
    double-negative
    DP
    double-positive
    E
    embryonic day
    Id
    inhibitor of DNA binding
    ILC
    innate lymphoid cell
    KO
    knockout
    PLZF
    promyelocytic leukemia zinc finger
    Tg
    transgenic.

  • Received August 12, 2013.
  • Accepted December 27, 2013.

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