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Blood, 1 May 2007, Vol. 109, No. 9, pp. 3757-3766. Prepublished online as a Blood First Edition Paper on January 16, 2007; DOI 10.1182/blood-2006-07-037655.
IMMUNOBIOLOGY Activation of the Hedgehog signaling pathway in T-lineage cells inhibits TCR repertoire selection in the thymus and peripheral T-cell activation1 Division of Cell and Molecular Biology, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, United Kingdom; 2 Section of Molecular and Cellular Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, United Kingdom; 3 Department of Immunology, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom
TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2  N2) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh–/– thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2N2 expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation.
The Hedgehog (Hh) family of secreted intercellular signaling molecules specifies cell fate and patterning during embryonic development and is involved in homeostasis and renewal of adult tissues, including skin, gut, lung, blood, and thymus.1–9
The specialized environment of the thymus supports the maturation of lymphocyte precursors into functional T cells through stages defined by cell surface expression of developmentally regulated markers: CD4–CD8– double-negative (DN) thymocytes differentiate to CD4+CD8+ double-positive (DP) cells that become mature CD4 or CD8 single-positive (SP) T cells. The DN population can be subdivided by expression of CD44 and CD25. The earliest CD44+CD25– cells (DN1) acquire CD25 (DN2), then lose CD44 expression (DN3) and become CD44–CD25–DN (DN4) cells before differentiating to DP. The transition from DN to DP cell requires a functional pre-T-cell receptor (TCR) complex,10 whereas maturation from DP to mature SP involves positive selection of the The 3 mammalian Hh proteins (Shh, Indian Hh, and Desert Hh) share a common signaling pathway. Hh proteins bind their cell surface receptor Patched, releasing the signal transducer Smoothened (Smo) to transmit the Hh signal into the cell. At the end of this pathway are the transcription factors Gli1, Gli2, and Gli3.1,2 Gli1 acts exclusively as an activator of transcription and, although not essential for mouse development or Hh signaling, is itself a Hh target gene, so measurement of its transcription can be used as a readout of Hh signaling in a given population of cells.13 Gli2 and Gli3 undergo processing to function as positive or negative regulators of transcription, depending on the presence or absence of Hh signaling, respectively.14 Both are essential for mouse development and have distinct but partially overlapping functions.15,16 Gli2 functions primarily as a transcriptional activator downstream of Hh signaling, and is necessary to initiate the first transcriptional changes induced by the Hh signal.17 In contrast, in vivo Gli3 functions primarily as a transcriptional repressor in the absence of the Hh signal.18 Analysis of Shh–/–, Gli3–/–, and Smo-deficient (conditional, T lineage) thymi has shown that Hh signaling is a positive regulator of the early stages of thymocyte development, controlling homeostasis of DN progenitors and differentiation from DN1 to DN2.5,7,8 Both the analysis of the Shh–/–5 and Gli3–/–7 thymus and in vitro experiments using wild-type mouse thymus explants4 have also suggested that Hh signaling is involved after TCR-ß gene rearrangement at the transition from DN to DP cell. Surprisingly, however, conditional deletion of Smo from T-lineage cells failed to show any influence of loss of Hh signaling after the DN2 stage.8 A role for Hh signaling has also been described in the activation of peripheral lymphocytes,19,20 although no defect in T-cell activation was detected in Smo-deficient lymphocytes.8 Here, we investigated the function of Hh signaling at late stages of thymocyte development and in T-cell activation. By analysis of the Shh–/– thymus, we show that Shh influences TCR repertoire selection and the transition from DP to SP. We also show that T-cell–autonomous activation of the Hh pathway in developing thymocytes inhibited TCR repertoire selection, altering differentiation to SP and the CD4/CD8 SP ratio. In addition, we demonstrate that in mature T cells the transcriptional changes induced by Hh reduce the ability of the TCR to signal for activation and proliferation, by a mechanism upstream of ERK phosphorylation.
Construction of lck-Gli2 N2 transgenic
Gli2 Mice
lck-Gli2 Genotyping
DNA was extracted using DNeasy tissue kit (Qiagen). Real-time polymerase chain reaction (PCR) for genomic Gli2 Cell sorting Thymocytes were sorted on a MoFlo (Cytomation, Fort Collins, CO). DN1-4 populations were sorted using antibodies against CD25FITC, CD3/4/8PE, and CD44Cychrome. Staining with CD3FITC, CD4PE, and CD8Cychome allowed sorting of DPs and SPs and CD8FITC, CD4PE, and B220Cychrome allowed sorting of peripheral SPs and B cells. Cells collected fell within FSC/SSC live gate.
Quantitative RT-PCR for Gli2
RNA was extracted using Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA) from fluorescence-activated cell sorter (FACS) cells. cDNA was synthesized using Superscript II (Invitrogen, Carlsbad, CA) and quantitative reverse-transcription (RT)–PCR carried out as described.7 Primers were Gli2 Antibodies and flow cytometry Cells were stained as described7 using directly conjugated antibodies from BD PharMingen (San Diego, CA) and analyzed on a FACScan (BD Biosciences, Basel, Switzerland). Live cells were gated according to FSC/SSC profiles. Data are representative of more than 3 experiments. CFSE (Sigma-Aldrich) labeling was carried out in PBS and 10 µM CFSE for 10 minutes at 25°C in the dark. DbSmcyPE tetramer (ProImmune, Oxford, United Kingdom) staining was prior to other antibody staining in PBS for 15 minutes at 25°C in the dark. In vitro T-cell activation Splenic T cells were cultured at 5 x 106/mL in AIM-V (Life Technologies), 10–5 M 2ME (Sigma-Aldrich) at 37°C, 5% CO2. Anti-CD3 and anti-CD28 (azide-free [NA/LE; BD PharMingen]) were at 0.01 µg/mL of each, unless otherwise stated. Fetal thymus organ culture (FTOC) FTOC was as described.4 Modified recombinant human Shh, as described,4 was a gift from Curis (Cambridge, MA). Analysis of Erk1 and Erk2 phosphorylation
Splenic T cells at 5 x 106 in OptiMEM (Invitrogen) media were activated with 1 µg/mL anti-CD3 For FACS analysis of intracellular phospho-Erk activation and staining were performed as described.27
Expression of Gli1 in SP thymocytes The Hh signaling pathway has previously been shown to be involved in regulation of thymocyte development at the transition from DN1 to DN25,7,8 and at the transition from DN to DP cell.4,5,7 Genes encoding components of the signaling pathway, including Smo and Gli1, are expressed by DN thymocytes, and anti-Smo staining and RT-PCR analysis of transcription in sorted DN subsets have both shown that their expression is highest in the DN2 subset.4,8 To assess activation status of the Hh signaling pathway at later stages of thymocyte development, we analyzed transcription of the Hh-target gene Gli1 in sorted thymocyte populations. We found that Gli1 transcription was down-regulated in the DN4 subset and DP population (Figure 1A). We did, however, detect an 11-fold increase in Gli1 transcription from the DP to CD4SP population, and a 4-fold increase from the DP to CD8SP population, indicating active Hh signaling at later stages of T-cell development, in SP cells (Figure 1A). The detection of Gli1 transcription in the CD4 and CD8 SP subsets is consistent with high cell surface expression of Smo on about 9% of CD4SP and 20% of CD8SP thymocytes.4 Shh is expressed by epithelial cells in the medulla and corticomedullary junction,4,6,8 and therefore expression of Gli1 and Smo in SP thymocytes is consistent with their location in the thymus, as like the early DN populations,28 they are located near the source of Shh.
Transgenic Gli2 expression in thymocytes
Given that the Hh signaling pathway is active in SP thymocytes, we investigated the function of Hh signaling at later stages of thymocyte development. We made a transgenic mouse that expressed an N-terminally truncated form of Gli2 under the control of the lck promoter. This truncated form of Gli2 (Gli2
Transgenic mice were produced and quantitative PCR of genomic DNA using primers that amplify both transgenic and genomic Gli2 gene demonstrated high copy number (Figure 1B). Quantitative RT-PCR demonstrated Gli2
Hh signaling in the Gli2
We analyzed lymphocyte populations in Gli2
Strength of TCR signaling can influence CD4/CD8 lineage commitment in the thymus, with a strong TCR signal favoring differentiation to CD4SP.31–34 We therefore analyzed expression of cell surface molecules associated with TCR signaling in thymocytes.27 Expression of CD235 was significantly increased on the DP population in the Gli2 N2 transgenic, from 59% in WT to 83% of DP cells in the Gli2N2 transgenic (Figure 2F), but was unaffected on SP populations. Expression of CD5, a molecule linked to attenuation of the TCR signal,36 was lower on DP and SP populations in the Gli2N2 transgenic compared with WT littermates (Figure 2G). In the CD8SP population, 72% stained brightly in WT compared with 45% in the Gli2N2 transgenic, and mean fluorescence intensity (MFI) was reduced from 113.9 in the WT to 80.9 in the Gli2N2 transgenic (Figure 2G). Expression of CD69, a marker associated with repertoire selection and T-cell activation, was higher on CD8SP thymocytes in the Gli2N2 transgenic, with, in a typical experiment, 74% staining positive compared with 60% in the WT (Figure 2H). Increased differentiation to CD4SP in the Shh–/– thymus Given that activation of the Hh signaling pathway in developing thymocytes reduced the ratio of CD4/CD8 SP thymocytes, we asked if removal of the Shh signal would have the opposing effect. Shh–/– embryos die before birth24 and have defects in DN thymocyte proliferation and differentiation, but in FTOC they accumulate a normal proportion of DP cells.5 We therefore cultured E14.5 Shh–/– and littermate thymi and analyzed the production of CD4SP cells. After 3 and 6 days in culture, the Shh–/– thymi contained a significantly greater proportion of CD4SP cells than those of littermates, indicating that Shh negatively regulates the transition from DP to CD4SP cell (Figure 3A-C). In E14.5+6-day FTOC, only 30% of SP cells express high cell surface CD3.5 Therefore to confirm that the increase in the CD4SP population was due to the accumulation of mature T cells, we also analyzed differentiation to SP cells in E17.5 Shh–/– and littermate FTOC after 7 days. On E17.5, the thymus contained approximately 70% DP cells and there was no significant difference in the proportion of DP cells between Shh–/– and littermate thymi. Mature SP cells had not arisen, facilitating investigation of the transition from DP to SP. In a typical experiment, the ratio of CD4/CD8 SP cells was increased from 2.6 in the Shh+/+ to 3.4 in the Shh–/– FTOC. The proportion of CD4SP cells was significantly increased from 13% in Shh+/+ FTOC to 21.2% in Shh–/– FTOC (Figure 3D). There was no difference in the proportion of CD3high cells in each SP population between Shh–/– and Shh+/+ littermate cultures, indicating that the increase in CD4SP cells was genuinely due to the accumulation of mature cells (Figure 3D). Of interest, CD69 expression was increased in the CD4SP Shh–/– population (Figure 3D). Therefore, under all culture conditions the Shh–/– thymus favored the production of CD4SP over CD8SP cells (Figure 3E).
When we compared the ratio of SP/DP in the Shh–/– thymus to that of littermates, we found an increased ratio under all culture conditions. Thus, despite the smaller size of the Shh–/– thymus,5 the DP population gave rise to relatively more SP cells (Figure 3F). Shh treatment reduced the CD4/CD8 ratio and CD5 expression in thymus explants
Given that the production of mature CD4SP cells was increased in the Shh–/– thymus, we asked if treatment of WT FTOC with recombinant Shh (r-Shh) could reduce differentiation to CD4SP. We treated E17.5 FTOC with r-Shh for 7 days. Typically, the percentage of DP cells in r-Shh–treated cultures increased to 78.5% compared with 70.7% in the control, whereas the proportion of CD4SP cells was decreased from 15% in control to 9.3% in treated cultures. When we compared the ratio of mature (CD3high) CD4/CD8 SP cells (Figure 4A), we found a dose-dependent reduction with maximum inhibition at 0.05 µg/mL r-Shh. CD5 expression was reduced in r-Shh–treated FTOC relative to controls in DP and SP populations (Figure 4B), consistent with the duller expression observed in the Gli2
When E17.5 FTOC were cultured for 10 days, Shh treatment again reduced differentiation from DP to CD4SP (Figure 4C). Gating on CD3high cells revealed a relative increase in the proportion of CD8SP cells from 14.9% in the control to 27.1% in Shh-treated cultures, concomitant with the decrease in the CD4SP population. Overall, the ratio of mature (CD3high) CD4/CD8 SP cells was reduced from 4.7 in the control to 2.1 in the Shh-treated cultures.
Taken together, these data show that Shh signaling is involved in regulating differentiation from DP to SP and CD4/CD8 ratio. Absence of Shh increased production of CD4SP cells, but Hh pathway activation, both by thymocyte autonomous Gli2
Negative selection is compromised in the Gli2
The pattern of expression of CD2, CD5, and CD69 in the Gli2
In the male HY-TCR transgenic model, negative selection in the thymus can commence at an earlier developmental stage than would occur physiologically because of early expression of the  ßTCR in DN cells.38 To confirm that inhibition of clonal deletion was not restricted to early TCR expression, we analyzed the effect of the Gli2N2 transgene on negative selection in male TCRß chain–only transgenic mice, B6.2.16ß.23 The B6.2.16ß mice express the same TCRß chain as the HY-TCR transgenic, but the TCR chain is variable and male-reactive DbSmcy-binding T cells are deleted in the male animals. The proportion of CD8SP cells (all of which expressed the transgenic ß chain) that stained brightly with DbSmcy tetramer in the lymph node was more than doubled in the presence of the Gli2N2 transgene (Figure 5C). Thus, clonal deletion of self-reactive thymocytes was also compromised in Gli2N2B6.2.16ß male mice. Hh signaling influences positive selection in HY-TCR transgenic female mice
In the female Gli2
In lymph nodes, the Gli2 Altered TCR repertoire selection in Shh mutant thymus To ask if Shh affects TCR repertoire selection in a physiological situation, we introduced the transgenic HY-TCR onto the Shh+/– background and compared positive selection in young adult female Shh+/– HY-TCR and their littermate thymi. We found increased positive selection of the transgenic TCR to the CD8 lineage in the Shh+/– HY-TCR thymus (Figure 6A) with an overall increase in the proportion of CD8SP thymocytes from 11% to 16%. When we gated on DbSmcybright cells (Figure 6A) the proportion of CD8SP cells was consistently increased, typically from 39% in the Shh+/+ littermate to 49% in the Shh+/– thymus, whereas the proportion of DP cells was decreased from 60% in the Shh+/+ to 49% in the Shh+/– thymus.
To analyze positive selection in the complete absence of Shh, we cultured female E17.5 Shh+/+ HY-TCR and Shh–/– HY-TCR littermate FTOC for 10 days. The proportion of HY-TCR+ CD8SP cells was increased approximately 3-fold (P = .027), from 8% in the Shh+/+ HY-TCR to 23% in the Shh+/– HY-TCR thymus (Figure 6B). Thus, in contrast to the Gli2 N2HY-TCR thymus, we found that in both the embryo and the adult, mutation of Shh increased positive selection of the HY-TCR. In the adult male HY-TCR thymus, clonal deletion is efficient and complete (Figure 5A and Kisielow et al22), and so we could not test if reduction in Shh copy number increased deletion. In the embryo, clonal deletion starts at E18 and a significant proportion of DP and CD8SP cells persists at birth and in the neonate.39,40 We therefore analyzed negative selection in the fetal and neonatal thymus.
In 12-day-old male Shh+/+ HY-TCR thymi, we detected a small population of HY-TCR+ CD8SP (
Attenuated T-cell activation in peripheral Gli2
Given that activation or reduction of Hh signaling altered the outcome of TCR ligation in thymocytes, we investigated the effect of Gli2
Proliferation of activated T cells is compromised by the Gli2 N2 transgene
To assess proliferation, splenocytes were labeled with CFSE and stimulated with anti-CD3 and anti-CD28. The Gli2 Hh signaling reduces TCR signal strength and inhibits TCR-induced ERK phosphorylation
To measure directly the effect of Hh signaling on TCR signals, we stimulated splenocytes from Gli2
Given that basal phospho-ERK levels were reduced in Gli2
Here, we show that the transcriptional events induced by activation of the Hh signaling pathway in mature peripheral T cells resulted in a reduction of TCR signal strength, profoundly inhibiting the ability of the TCR to transduce signals for activation and proliferation. Likewise, activation of the Hh signaling pathway in thymocytes changed the outcome of TCR ligation at many stages of development: allowing self-reactive T cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection of CD8 T cells; and reducing the ratio of CD4/CD8 SP cells produced in the thymus. In contrast, in the Shh–/– thymus the CD4/CD8 ratio was affected in the opposite way, and differentiation from DP to CD4SP was increased. Both positive and negative selection of a transgenic TCR were also increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from DP to SP cell in a physiological situation. Taken together, the consequences for thymocyte development of removal of the Shh signal (in the Shh–/– thymus), or activation of Hh signaling in vivo (by Gli2 N2 expression) and in vitro (by Shh treatment), all strongly support the hypothesis that Hh signaling in thymocytes modulates TCR signal strength. Reduction of TCR signal strength by Hh-dependent transcription has many consequences for thymocyte development and peripheral immunity, as the outcome of TCR ligation would depend on the Hh microenvironment of the cell. Hh proteins have been shown to function as morphogens,1,41 produced at localized sources and specifying different cell fates in a concentration-dependent manner. Thymocytes move through distinct areas of the thymus at different stages of their development,28 and therefore will receive different strengths of Hh signal at different stages. Our data suggest that their location in the thymus, relative to the source of Hh, will influence the outcome of TCR ligation (positive or negative selection, CD4/CD8 lineage decisions). Of interest, Shh is expressed by epithelial cells scattered in the medulla and corticomedullary junction, and the thymocyte populations that are located in this part of the thymus (early DN populations and SPs) show highest expression of the Hh-target gene Gli1 (Figure 1). As not all medullary epithelial cells express Shh,4,6 it is possible that those that express Shh are specialized for a particular function in TCR repertoire selection. In the periphery, Hh signaling could function to change the threshold of T-cell activation and influence TCR-dependent T-cell fate decisions, such as TH1/TH2 differentiation or the induction of anergy. Shh is expressed by follicular dendritic cells,42 but Hh proteins are also expressed in skin, gut, and lung, where they are involved in tissue homeostasis and renewal.1 These tissues as sites of frequent immune challenge by pathogens are also subject to many inflammatory and autoimmune diseases, and Hh expression could function to reduce the initial threshold for T-cell activation. Up-regulation of Hh proteins during renewal or remodeling after infection or tissue damage could further influence T-lymphocyte populations at sites of damage.
Inappropriate activation of the Hh signaling pathway has been implicated in many cancers,2 and expression of Gli2
Inhibition of TCR signaling by Hh-induced transcriptional changes involves compromised MAPKinase activation (Figure 7E-G). It is unclear why addition of IL-2 restores proliferation of activated Gli2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
and anti-CD28 antibodies and incubated on ice for 30 minutes. For stimulation, cells were transferred to 37°C for 5, 15, 30, 45, and 60 minutes in 5% CO2. After a short spin, cells were lysed on ice for 40 minutes and analyzed by immunoblotting using anti-Erk1, anti-Erk2, anti–phospho-Erk, and anti–phospho-Erk2 (Cell Signaling, Beverly, MA) as described.
, n = 6). (B) CD4 and CD8 profiles in the Gli2
) and Gli2
3%) of peripheral lymph node cells expressed high levels of CD8 and stained brightly with DbSmcy tetramer in the Gli2
