Differential Roles of Cytokine Receptors in the Development of Epidermal γδ T Cells¹

Development of γδ T cells has unique features in contrast to αβ T cell development (1, 2, 3). γδ T cells expressing a specific V γ-chain appear as several successive waves in the developing thymus, and each of them shows distinct tissue distribution in the adult mouse. Vγ3⁺ T cells develop as the first wave in the fetal thymus and become Thy-1⁺ dendritic epidermal T cells (DETC)⁴ in the adult skin. Vγ4⁺ T cells are the second wave and are distributed in the epithelium of the lung, tongue, and vagina. These two epithelial T cell subsets express homogeneous γδ TCRs, Vγ3/Vδ1 or Vγ4/Vδ1, with a single species of Vγ-Jγ junctional sequence. Vγ2⁺ T cells develop in the thymus after these cells and reside mainly in the spleen and the lymph nodes. In contrast, the majority of γδ intestinal intraepithelial lymphocytes (IEL) express Vγ5 TCR and develop in the gut from cryptopatches (4, 5). This sequential development of γδ T cells is regulated by the V-J recombination of the TCRγ locus (6) and is predetermined at the level of hematopoietic stem cells (7, 8).

IL-7 is an essential cytokine for γδ T cell development. IL-7 exerts its effect through interaction with the IL-7R, which is composed of a unique α-chain (IL-7Rα) and the common cytokine receptor γ-chain (γ_c) (9). Recently, thymic stromal lymphopoietin has been shown to transmit signals through IL-7Rα and thymic stromal lymphopoietin receptor heterodimer (10, 11). Injection of neutralizing Abs to IL-7 or IL-7Rα or genetic ablation of IL-7, IL-7Rα, or γ_c leads to a block of lymphocyte development (12, 13, 14, 15, 16, 17). Although IL-7Rα-deficient mice have small numbers of B cells and αβ T cells in periphery, they totally lack γδ T cells (18, 19). The IL-7R transmits at least two types of signals in lymphocyte progenitors (20). One signal is for survival and proliferation. For instance, the IL-7R induces the expression of Bcl-2 in T cell precursors (21), and introduction of Bcl-2 transgenes partly restores αβ T cell development in IL-7Rα-deficient mice (22, 23). The IL-7R supports the proliferation of lymphocyte precursors through the activation of phosphatidylinositol (PI)-3 kinase (24, 25). Peripheral αβ T cells in IL-7R-deficient mice are also defective in survival and proliferation (26). The second signal from the IL-7R is to promote recombination and transcription in the IgH and TCRγ loci. For example, IL-7R signaling induces germline transcription and DNA rearrangement in D-distal V segments in pro-B cells (27). The V-J recombination and transcription of TCRγ genes is also severely impaired in IL-7Rα-deficient mice (28, 29, 30). STAT5, a signaling molecule of the IL-7R, induces germline transcription in the TCRγ locus and promotes Vγ-Jγ recombination and γδ T cell development (31).

In additon to IL-7, other cytokines play substantial roles in γδ T cell development. IL-15 achieves its effects through interaction with the IL-15R, which is composed of a unique α-chain (IL-15Rα), the common β-chain (IL-2Rβ), and the γ_c (32). Deletion of IL-15, IL-15Rα, or IL-2Rβ results in a block of NK cell development and impaired γδ T cell development in the epithelium (33, 34, 35). Mature Vγ3⁺ thymocytes in the fetal thymus express IL-2Rβ, and in utero treatment with anti-IL-2Rβ Ab abrogates DETC in the adult skin (36). IL-2Rβ-deficient mice have decreased numbers of γδ IEL (33). These mice also have defective maturation of Vγ3 thymocytes and lack DETC (37). These results suggest that IL-15 plays important roles in the development of γδ IEL and DETC. Besides IL-15, IL-2 is also implicated in thymic and extrathymic T cell development (38).

Both the IL-7R and the IL-2/IL-15R mobilize a similar set of signaling molecules (9). The ligand binding to the receptors triggers the phosphorylation and activation of receptor-associated Janus kinase (Jak)1 and Jak3 tyrosine kinases. Following their activation, the Jak kinases phosphorylate the specific tyrosine residues of IL-7Rα and IL-2Rβ. The STAT proteins, and PI3 kinase in case of the IL-7Rα, are recruited to the tyrosine residues and subsequently phosphorylated and activated by the Jak kinases. The receptors mainly activate STAT5A and STAT5B. The phosphorylated STAT5 proteins then form dimers and bind to a consensus motif and activate the transcription of various target genes, including TCRγ genes. PI3 kinase phosphorylates PI and activates Akt. Akt protein in turn inactivates Bad, caspase-9, and IκB kinase-α proteins by phosphorylation and transmits antiapoptotic signals. The Jak kinases also activate signal-transducing adaptor molecule and proline-rich tyrosine kinase-2 proteins. This signaling pathway then activates mitogen-activated protein kinase cascade and results in cell proliferation. The IL-7R also induces Bcl-2 expression in early T cells by an unidentified pathway (21).

The roles for each signaling pathway in γδ T cell development are largely unknown. Introduction of a Vγ1.1/Vδ6 TCR γδ transgene failed to restore γδ T cells in γ_c-deficient mice, suggesting that the IL-7R plays some role in proliferation and/or survival of γδ T cells (39). Introduction of a Bcl-2 transgene partly restored αβ, but not γδ, T cell development in IL-7Rα-deficient mice (31). Similarly, the Bcl-2 transgene alone did not rescue B cells, γδ T cells, and NK cells in γ_c-deficient mice (40). However, these experiments did not clarify the role of Bcl-2 in γδ T cell development, because the IL-7R signaling is necessary for the recombination and transcription of TCRγ genes. To clarify the roles of the cytokine receptors in the development of epidermal γδ T cells, we introduced a Vγ3/Vδ1 TCR γδ transgene into IL-7Rα-deficient mice and found that they restored Vγ3⁺ T cells in the fetal thymus and the skin. In contrast, the same Vγ3/Vδ1 TCR transgene failed to rescue γδ T cells in the skin of IL-2Rβ-deficient mice. Further addition of a Bcl-2 transgene did not recover epidermal γδ T cells in IL-2Rβ-deficient mice. Thus, this study revealed that IL-7R and IL-2/IL-15R serve differential functions in epidermal γδ T cell development.

IL-7Rα-deficient (18), IL-2Rβ-deficient (41), and H-2K-Bcl-2-transgenic (Tg) mice (42) were reported previously and were bred on the (129/Ola × C57BL/6) hybrid background. The Bcl-2 transgene is driven by a H-2K promoter and expressed on virtually all blood cells. Tg mice containing productively rearranged Vγ3/Vδ1 TCR genes were described before (43) and maintained on the C57BL/6 background. The Vγ3/Vδ1 TCR is derived from a DETC line. The genotype of mice and fetuses was determined by PCR. The age of fetuses was determined by taking the appearance of a vaginal plug as day 0. All mice were maintained under the specific pathogen-free conditions in the Institute of Laboratory Animals at the Graduate School of Medicine, Kyoto University (Kyoto, Japan).

Thymocytes and spleen cells were harvested in PBS supplemented with 2% FBS and 0.02% sodium azide (FACS solution). RBCs were lysed, and cells were washed in the FACS solution. Epidermal cells were isolated from the ears as previously described but with slight modifications (44). Briefly, the ears were separated into two sides with fine forceps and incubated on PBS containing 1% trypsin for 45 min at 37°C. Epidermal sheets were separated from the dermis with fine forceps. Epidermal cells were released by gently rubbing the sheets with a plastic rod. The viable cells were purified by density gradient centrifugation with Lympholyte-M (Cedarlane Laboratories, Hornby, Onatrio, Canada) and cultured in RPMI 1640 medium containing 10% FBS and 5% conditioned medium of Con A-stimulated rat spleen cells for 24 h.

The following mAbs were used: FITC-conjugated Abs 145-2C11(anti-CD3ε), 53-6.7 (anti-CD8α), H57-597 (anti-αβ TCR), 536 (anti-Vγ3/Vδ1 TCR), M1/69 (anti-CD24), and 53-2.1 (anti-Thy-1.2); biotin-conjugated Abs H57-597 (anti-αβ TCR) and M181.1 (anti-Vγ3/Vδ1 TCR); and PE-conjugated Abs GK1.5 (anti-CD4), GL3 (anti-γδ TCR), and 2G9 (anti-I-A^d/I-E^d). The above-mentioned mAbs were purchased from BD PharMingen (San Diego, CA). Biotin-anti-Vγ3/Vδ1 TCR Ab was a gift from Dr. I. MacNeil (Ariad Pharmaceuticals, Cambridge, MA) (7), and PE-streptavidin was obtained from Biomedia (Foster City, CA). Flow cytometric analysis was performed as previously described (31). Debris, erythrocytes, and dead cells were excluded from the analysis by forward and side scatters and propidium iodide gatings. Viable cells were analyzed by a FACSCalibur with CellQuest software version 3.1 (BD Biosciences, San Jose, CA).

Immunofluorescence staining of epidermal sheets was done as described previously (44). Briefly, the sheets were fixed in cold acetone for 20 min, rinsed in PBS, and incubated with FITC-anti-Thy-1.2 or FITC-anti-Vγ3/Vδ1 TCR Ab for 2 h at room temperature. After rinses in PBS, the sheets were mounted on slide glass and examined under a fluorescence microscope.

To test the role of the IL-7R in the development of epidermal γδ T cells, we introduced a Vγ3/Vδ1 TCR transgene into the IL-7Rα-deficient mice to bypass the defective Vγ-Jγ recombination (28, 29). We backcrossed the Vγ3/Vδ1 TCR-Tg mice to IL-7Rα^−/− mice and chose four genotypes of mice, namely, IL-7Rα^+/−, Vγ3/Vδ1 TCR-Tg⁺IL-7Rα^+/−, IL-7Rα^−/−, and Vγ3/Vδ1 TCR-Tg⁺IL-7Rα^−/−. Thymocytes and spleen cells were isolated from the mice and analyzed by flow cytometry (Fig. 1). Although only 0.3% of thymocytes expressed γδ TCR in IL-7Rα^+/− mice, 14% of thymocytes were γδ T cells expressing the Vγ3/Vδ1 TCR transgene in TCR-Tg⁺IL-7Rα^+/− mice. In TCR-Tg⁺IL-7Rα^+/− mice, 51% of thymocytes were CD4⁻CD8⁻, suggesting that many of the Tg γδ T cells were CD4⁻CD8⁻. In contrast, we did not detect any distinct TCRγδ⁺ cells in IL-7Rα^−/− mice, as we and others reported previously (18, 19). In TCR-Tg⁺IL-7Rα^−/− mice, 6.3% of thymocytes were γδ T cells expressing the Vγ3/Vδ1 TCR transgene. These results suggest that the introduction of the Vγ3/Vδ1 TCR transgene alone significantly rescued γδ T cell development in the thymus of IL-7Rα^−/− mice. In contrast, introduction of the Vγ3/Vδ1 TCR transgene gave rise to much poorer recovery of Vγ3⁺ T cells in the spleen of IL-7Rα^−/− mice (Fig. 2). A small but distinct Vγ3⁺ T cell population (0.95–2.00% in four mice) constantly developed in the spleen of TCR-Tg⁺IL-7Rα^+/− mice, although some double-positive signals (0.70–1.70%) were detected, partly due to the appearance of TCRγδ^dull αβ T cells by the effect of the transgene. In contrast, no Vγ3⁺ T cell populations were detected in IL-7Rα^−/− and TCR-Tg⁺IL-7Rα^−/− mice.

Next we compared the overall numbers of total and γδ T cells in the thymus (Fig. 3). The numbers of γδ T cells dramatically increased in the thymus of TCR-Tg⁺IL-7Rα^+/− mice compared with IL-7Rα^+/− mice. The introduction of the TCRγδ transgene also resulted in a significant increase of γδ T cells in the thymus of IL-7Rα^−/− mice. These results confirmed that the introduction of the Vγ3/Vδ1 TCR transgene alone partly rescued γδ T cell development in the thymus of IL-7Rα^−/− mice. According to a previous study (30), our Vγ3/Vδ1 TCR transgene seems to be classified into “high copy type,” which gives rise to substantial recovery of γδ T cells in IL-7Rα^−/− mice.

To test the role of the IL-7R on cell survival of γδ T cells, we next introduced an H-2K-Bcl-2 transgene into the Vγ3/Vδ1 TCR-Tg⁺IL-7Rα^−/− mice. Introduction of the Bcl-2 transgene had a minimal effect on the numbers of γδ T cells in the thymus of TCR-Tg⁺IL-7Rα^−/− mice (Fig. 3). The numbers of γδ T cells in TCR-Tg⁺Bcl-2-Tg⁺IL-7Rα^−/− mice did not reach those in TCR-Tg⁺IL-7Rα^+/+ mice. Therefore, these results suggest either that γδ T cells depend on IL-7R signals mainly for their proliferation in the thymus, or that the impaired transcription of TCR γ genes caused by IL-7R deficiency may still have an adverse effect on γδ T cell development.

Because Vγ3⁺ T cells develop as the first wave of γδ T cells in the fetal thymus, we checked E17 fetal thymocytes by flow cytometry (Fig. 4). In IL-7Rα^+/+ mice, 1.9% of thymocytes were Vγ3⁺. Some of them were Vγ3/Vδ1 TCR⁺CD24⁻, suggesting they were at mature stages (45). However, in IL-7Rα^−/− fetal thymus, few cells expressed Vγ3/Vδ1 TCR, in agreement with the previous observation that IL-7Rα-deficient mice lack γδ T cells in the fetal thymus (18). On the contrary, 40% of thymocytes were Vγ3⁺ T cells in TCR-Tg⁺IL-7Rα^−/− fetuses. A part of these Tg γδ T cells were at mature stages expressing Vγ3/Vδ1 TCR⁺CD24⁻.

Next we compared the overall numbers of total and Vγ3⁺ fetal thymocytes (Fig. 5). TCR-Tg⁺IL-7Rα^−/− fetuses had increased numbers of total and Vγ3⁺ thymocytes compared with IL-7Rα^−/− fetuses, suggesting that the expression of the TCR transgene promoted γδ T cell development in IL-7Rα-deficient fetal thymus. Even compared with normal IL-7Rα^+/+ fetuses, TCR-Tg⁺IL-7Rα^−/− thymus contained 7-fold more Vγ3⁺ T cells. These results showed that Vγ3⁺ T cells can normally expand in the fetal thymus without the IL-7R after they express the TCR. Thus, the results implied that Vγ3⁺ T cells may depend on other cytokine(s), such as IL-15, for their expansion in the fetal thymus. They also suggest that IL-7R is essential for V-J recombination of TCRγ genes.

Because these Vγ3⁺ T cells are distributed as DETC in the skin of adult mice (46), we next isolated epidermal cells from IL-7Rα^+/−, TCR-Tg⁺IL-7Rα^+/−, IL-7Rα^−/−, and TCR-Tg⁺IL-7Rα^−/− mice and analyzed them by flow cytometry (Fig. 6). Vγ3/Vδ1 TCR⁺ DETC were detected in both IL-7Rα^+/− and TCR-Tg⁺IL-7Rα^+/− mice. IL-7Rα^−/− mice lacked these cells, in agreement with the previous observation that IL-7Rα-deficient mice lack DETC (18). However, TCR-Tg⁺IL-7Rα^−/− mice contained abundant DETC expressing Vγ3/Vδ1 TCR in the skin. We equally detected MHC class II⁺ Langerhans cells in all of these mice. Similar results were obtained by fluorescence staining of epidermal sheets (data not shown). These results suggest that the IL-7R is probably dispensable for the maintenance of DETC in the skin.

Because IL-2Rβ-deficient mice have decreased numbers of mature Vγ3⁺ thymocytes and lack DETC (37), the IL-2Rβ plays an essential role in the development of Vγ3⁺ T cells in the fetal thymus and the skin. To test the role of the IL-2Rβ in proliferation and survival of Vγ3⁺ T cells, we introduced the Vγ3/Vδ1 TCR transgene into IL-2Rβ-deficient mice to bypass Vγ-Jγ recombination and to help keep the expression of the TCR. We first analyzed adult thymocytes by flow cytometry (Fig. 7,A). There was no significant difference in Vγ3⁺ T cells between TCR-Tg⁺IL-2Rβ^+/− and TCR-Tg⁺IL-2Rβ^−/− mice. The numbers of Vγ3⁺ thymocytes were also unchanged (Fig. 7 B). These results suggest that IL-2Rβ is dispensable for expansion of Vγ3⁺ T cells in the adult thymus.

To assess the role of the IL-2Rβ in DETC development, we next stained epidermal sheets with either anti-Thy-1.2 or anti-Vγ3 TCR Ab (Fig. 8). We detected abundant DETC in the skin of TCR-Tg⁺IL-2Rβ^+/− mice. In contrast, TCR-Tg⁺IL-2Rβ^−/− mice totally lacked DETC. These results supported the idea that the IL-2/IL-15R plays an essential role either in maturation of Vγ3⁺ T cells in the fetal thymus or in expansion and/or survival of DETC in the skin (37). Introduction of the additional Bcl-2 transgene failed to restore DETC in TCR-Tg⁺IL-2Rβ^−/− mice. Therefore, the forced expression of Bcl-2 is not enough to substitute for IL-2Rβ signals in DETC development, suggesting that IL-2/IL-15R probably transmits an active proliferation signal for DETC in the skin.

In this study, we first showed that introduction of the Vγ3/Vδ1 TCR transgene alone partly rescued γδ T cell development in the thymus, but not in the spleen, of IL-7Rα^−/− mice ( Figs. 1–3). Introduction of the Bcl-2 transgene in these mice had a minimal effect on γδ T cells in the thymus (Fig. 3), suggesting that the IL-7R mainly transmits proliferation signals for γδ T cells in the thymus. In contrast to the adult thymus, the introduction of a Vγ3/Vδ1 TCR transgene into IL-7Rα^−/− mice completely restored Vγ3⁺ T cells in the fetal thymus and DETC in the adult skin ( Figs. 4–6). The IL-2/IL-15R probably substituted for the IL-7R in supporting expansion of Vγ3⁺ thymocytes after the expression of the TCR. In contrast, the same Vγ3/Vδ1 transgene alone or together with the Bcl-2 transgene failed to rescue DETC in the skin of IL-2Rβ^−/− mice (Fig. 8). These results suggest that the IL-2/IL-15R, rather than the IL-7R, plays an essential role in proliferation and survival of DETC in the skin. Thus, this study proved that the IL-7R and the IL-2/IL-15R serve differential functions in epidermal γδ T cell development.

The IL-7R transmits at least two signals during γδ T cell development. One is for proliferation and survival, and the other is for recombination and transcription of the TCRγ locus. As we and others previously showed, the V-J recombination of TCRγ genes was severely impaired in IL-7Rα-deficient mice (28, 29). In addition, STAT5, a signaling molecule of the IL-7R, induced germline transcription in the TCRγ locus and promoted Vγ-Jγ recombination and γδ T cell development (31). In this study, the Vγ3/Vδ1 transgene completely rescued DETC in the fetal thymus and the skin of IL-7Rα-deficient mice. These results suggest that the IL-7R is essential for recombination of TCRγ genes even in the fetal thymus. This is probably because the IL-2/IL-15R is not expressed at very early stages when V(D)J recombination takes place.

Thymic γδ T cell development depends on IL-7R signaling for both proliferation and survival. In previous reports, the IL-7R induced the expression of Bcl-2 in T cell precursors (21), and introduction of a Bcl-2 transgene partly restored αβ T cell development in IL-7Rα-deficient mice (22, 23). This is probably because αβ T cell precursors receive proliferation and survival signals from pre-TCR after they manage to express TCR β-chain (47). In contrast, γδ T cell precursors seem to depend entirely on the IL-7R for their proliferation and survival in the thymus. The Bcl-2 transgene had a minimal effect on γδ T cells in the thymus of Vγ3/Vδ1 TCR-Tg⁺IL-7Rα^−/− mice (Fig. 3), in accordance with the previous reports that Bcl-2 transgenes did not rescue γδ T cells in IL-7Rα^−/− and γ_c^−/− mice (31, 48). Therefore, γδ T cell precursors receive proliferation and survival signals from the IL-7R mainly irrespective of Bcl-2. This can be mediated by a carboxyl-terminal region of the IL-7Rα through the activation of PI3 kinase and Pim-1 (25, 49, 50). It is also conceivable that the IL-7R supports the survival of γδ T cells by keeping the transcription of the TCRγ genes (30). Because the TCR transgene expression on recovered γδ T cells was not lowered, our results suggest that impaired expression of the transgene alone would not explain the defective development of γδ T cells in these mice.

Extrathymic γδ T cell development depends not only on the IL-7R but also on the IL-2/IL-15R. Although IL-7Rα-deficient mice completely lack γδ IEL (18), IL-15-, IL-15R-, and IL-2Rβ-deficient mice show only decreased numbers of γδ IEL (33, 34, 35). Because a Vγ2/Vδ5 TCR transgene did not completely rescue γδ IEL in IL-7Rα-deficient mice, it is implied that the IL-7R promotes proliferation and/or survival of γδ IEL besides inducing recombination of TCRγ genes (S.-K. Ye and K. Ikuta, unpublished data). In addition, γδ IEL probably receive proliferation and survival signals from the IL-2/IL-15R in response to IL-15 produced by intestinal epithelial cells. Thus, the IL-7R and the IL-2/IL-15R play their roles at early and late stages of γδ IEL development, respectively.

DETC development has unique features. The Vγ3/Vδ1 TCR transgene completely rescued Vγ3⁺ DETC in the fetal thymus and the skin of IL-7Rα-deficient mice. In contrast, the same transgene failed to restore DETC in the skin of IL-2Rβ-deficient mice. These results supported the idea that the IL-2/IL-15R plays an essential role either in maturation of Vγ3⁺ T cells in the fetal thymus or in expansion and/or survival of DETC in the skin (37). Because IL-2Rβ-deficient mice had only decreased numbers of mature Vγ3⁺ T cells in the fetal thymus, the main reason for the lack of DETC in Vγ3/Vδ1⁺IL-2Rβ^−/− mice would be that DETC and their precursors depend mostly on the IL-2/IL-15R for their proliferation and survival in the skin. Because the Bcl-2 transgene did not restore DETC in the skin of Vγ3/Vδ1⁺IL-2Rβ^−/− mice, DETC are likely to receive a proliferation signal from the IL-2Rβ even in the skin. These results are consistent with our previous results that exogenous addition of IL-15 to organ culture of fetal skin induced proliferation of Vγ3 DETC (37).

We thank Drs. M. Iwashima, S. Sakaguchi, I. MacNeil, J. Domen, K. Akashi, and I. L. Weissman for materials and discussion; M. Iidaka, M. Sugimori, Y. Kobayashi, T. Taniuchi, and M. Tanaka for their excellent technical assistance; and Dr. S. Takeda for critically reading the manuscript.