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The centrosome is essential for cytotoxic T lymphocyte (CTL) function, contacting the plasma membrane and directing cytotoxic granules for secretion at the immunological synapse. Centrosome docking at the plasma membrane also occurs during cilia formation. The primary cilium, formed in non-haemopoietic cells, is essential for vertebrate Hedgehog (Hh) signaling. Lymphocytes do not form primary cilia, but here we found that Hh signaling played an important role in CTL killing. TCR activation, which “pre-arms” CTL with cytotoxic granules, also initiated Hh signaling. Hh pathway activation occurred intracellularly and triggered Rac1 synthesis. These events “pre-armed” CTL for action by promoting the actin remodelling required for centrosome polarisation and granule release. Thus Hh signaling plays a role in CTL, and the immunological synapse may represent a modified cilium.
Cytotoxic T lymphocytes (CTL) recognise tumor and virally infected cells via their T cell receptor (TCR). Recognition triggers a cascade of intracellular signaling that leads to the formation of the immunological synapse and polarisation of the centrosome to contact the plasma membrane (1) at the central supramolecular activation complex (cSMAC) (2) where TCR clusters within the synapse (1, 3). Cytotoxic granules move towards the docked centrosome and deliver their contents precisely at the point of TCR-mediated recognition, focussing secretion towards the target cell to be destroyed. Docking of the centrosome also occurs during cilia formation, when the mother centriole contacts the plasma membrane, forming the basal body from which the cilium extends. Although lymphocytes are one of very few cell types that do not form primary cilia (4) morphological and functional similarities can be drawn between the immunological synapse and cilia. Endocytosis and exocytosis are focussed at the point of centrosome docking in both cases (5); ciliary intraflagellar transport (IFT) proteins are found in T cells (6), and both structures form important signaling platforms (1, 2, 7, 8).
In Hh signaling, binding of exogenous Sonic, Indian or Desert Hh (Shh, Ihh, Dhh) to the transmembrane receptor Patched (Ptch) regulates translocation of Smoothened (Smo) to primary cilia (9, 10). The ciliary localisation of Smo is required to initiate transduction of Gli-mediated transcription of target genes, including Gli1, which serves as a reporter of Hh signaling (7, 8). We asked whether proteins of the Hh pathway are expressed in T cells and whether TCR activation triggered Hh signaling. Naïve CD8 T cells and CTL derived after 4-5 days of in vitro TCR activation were isolated from OT-I TCR transgenic mice. TCR cross-linking was triggered in both naïve CD8 T cell and CTL populations using plate-bound anti-CD3 antibody (Fig.1A). In naïve CD8 T cells Gli1 mRNA was not detected, but expression was induced upon TCR cross-linking, peaking at 12h. Controls lacking anti-CD3 showed no Gli1 expression. CTL had low Gli1 mRNA levels that increased 180-fold after TCR ligation (Fig. 1A). In addition the genes encoding Ptch1 and 2 receptors the signal transducer Smo, and the ligand Ihh were all expressed in both naïve CD8 and CTL (Figure S1A), and protein expression of Ptch, Gli1 and Ihh increased after TCR activation of naïve CD8 cells (Fig. 1B) and CTL (Figure S1B). Neither Shh nor Dhh were detected in CD8 T cells before or after 24h TCR activation, or EL4 and P815 target cell lines, (Fig. S1C). When TCR signaling was severely impaired by deletion of the upstream tyrosine kinase Lck (11), induction of Gli1 was also diminished in naïve CD8 T cells (Fig. 1C,D). Thus CD8 T cells express Hh pathway components and require TCR signaling to trigger Hh signaling.
Because only T cells were present in these assays, CD8 T cells must both have synthesised and responded to Hh proteins to activate this signaling pathway. This is unusual as Hh signaling is usually paracrine, with one cell type producing Hh and another responding to this cue. We noted that Ihh was detected as a 45kDa protein, indicating that it was not fully processed into the secreted form (12, 13). This raised the possibility that Ihh might bind Ptch intracellularly. We used recombinant Ihh protein to ask whether CTL responded to exogenous Ihh. Although cross-linking of TCR triggered Gli1 expression, stimulating CTL with extracellular Ihh alone did not. Furthermore exogenous Ihh did not enhance Gli1 expression in response to TCR activation (Fig. S1E). Thus Ihh encounters its receptor, Ptch, intracellularly in CTL.
We next asked where Ptch, Ihh and Smo localise in CTL using antibodies to detect endogenous Ihh and endogenous Smo, and Ptch1-YFP to localise the receptor (Fig. 2). In CTL, Ptch1 was found on intracellular vesicles (Fig.2A). Ihh co-localised with Ptch1 on a subset of these vesicles, which polarised towards the immunological synapse upon recognition of a target cell (Fig. 2B, C). This is consistent with the idea that Ihh-mediated signaling via Ptch1 takes place intracellularly, and we confirmed the interaction between Ihh and Ptch1 on intracellular vesicles using a proximity ligation assay (Fig. S2A). Ihh was also seen near sites of actin accumulation (Fig. S2B,C), raising the possibility that Ihh may also influence actin as shown for other Hh components (14, 15). Endogenous Smo was also associated with intracellular vesicles in CTL (Fig. 2D) that are predominantly Lamp1-positive (Fig. S2D) and distinct from the Ihh-positive compartment (Figure S2E). Live imaging of Smo-EGFP expressed in CTL showed that, upon recognition of a target cell via TCR, the intracellular pool of Smo polarised to the immunological synapse (Fig. 2E and Movie 1), analogous to the Hh-triggered translocation of Smo into the cilium (9, 10, 16, 17).
To determine whether TCR-triggered Hh signaling affects CTL-mediated killing, we made use of a genetic model in which Smo is conditionally deleted. Although Hh signaling is required for T cell development (18-21), T cells from mice, in which exon 1 of Smo is inducibly deleted in adult haemopoietic cells under Mx1-Cre control, develop normally (22, 23). CTL generated from Smo-deleted mice showed over 65-fold reduced levels of Smo mRNA relative to controls and greatly reduced Smo protein levels, (Fig. 3A, B). Gli1 mRNA upregulation in response to TCR ligation was also reduced: CTL from control mice showed a 3-fold increase in levels of Gli1 mRNA, whereas CTL from Smo-deleted mice gave only a 1.5-fold increase (Fig. 3A). TCR signaling was not altered in Smo-deleted CTL (Fig. 3C and S3). However when we assessed Smo-deleted CTL for their cytotoxic effector function we found reduced levels of target cell killing compared to control CTL (Fig. 3D) suggesting that Hh signaling contributes to CTL killing. Because centrosome polarisation to the plasma membrane is a key step in CTL mediated killing, we asked whether Hh signaling affected centrosome docking at the cSMAC during conjugate formation between CTL and target cells. Smo-deleted CTL showed a ~50% reduction in centrosomal docking at the cSMAC (Fig. 3E) consistent with the reduction in Smo protein levels and CTL killing.
We also confirmed that Hh signaling is important for centrosome polarisation and CTL-mediated killing using three separate inhibitors: cyclopamine and vismodegib (GDC-0449) both of which inhibit Smo (24, 25) and GANT61 which targets Gli transcription factors (26). The inhibitors reduced levels of Gli1 and Ptch protein, both targets of the Hh signaling pathway, but did not affect levels of TCR-associated kinases Lck and ERK or granzyme A and perforin, two CTL proteins required for target cell lysis (Fig. 3F, S4A). TCR signaling was also unaffected by these inhibitors (Fig. 3G, S4B). All three inhibitors diminished CTL-mediated killing in a dose-dependent manner (Fig. 3H, S4C), without impairing conjugate formation with target cells or clustering of Lck at the cSMAC in response to TCR signaling (Fig. S4E, F). Centrosome docking at the cSMAC was also reduced in conjugates formed by CTL treated with inhibitor (Fig. 3I).
Centrosome polarisation has been correlated with actin remodelling at the immunological synapse (1, 3). Carrier treated CTL reorganised actin into a distal ring at the immunological synapse, and polarised the centrosome within 5 minutes of encountering the target (Fig. S5A and Movie S2). By contrast in cyclopamine-treated CTL, actin accumulated across the immunological synapse (t=0), but failed to reorganise into the distal actin ring (Fig. S5B). Centrosome polarisation to the plasma membrane was also disrupted (Movie S3): 92% of conjugates polarised the centrosome to the synapse in control carrier-treated CTL compared with 59% of conjugates in cyclopamine-treated CTL. Actin clearance from the immunological synapse was also greatly reduced in CTL from Smo-deleted mice (Fig. 4A-C), and Smo-deleted CTL showed a 60% reduction in actin clearance from the synapse compared with control CTL. Thus Hh signaling might also be required to promote actin reorganisation at the immunological synapse.
Centrosome polarisation in T cells is driven by a process of microtubule end-on capture-shrinkage, in which microtubules emanating from the centrosome are captured at the cortex and then undergo shrinkage. Both this process and actin remodelling are mediated by Rac1 (27-30). Furthermore the Hh pathways has been implicated in Rac1-mediated actin remodelling in neurons and fibroblasts (14, 15). Therefore we examined Rac1 expression during induction of CTL from naïve T cells. Rac1 protein levels increased after TCR stimulation of naïve T cells (Fig. 4D). By contrast both protein and mRNA levels of Rac1 were diminished in Smo-deleted CTL (Fig. 4E,F), suggesting that Rac1 levels were regulated by Hh signaling via a transcriptional effect.
These findings support a model for the regulation of CTL-mediated killing by Hh signaling (Fig. S5C). Naive T cells are small, round cells lacking both the cytotoxic granules and the highly developed cytoskeleton required for target cell killing. Upon TCR activation these cells develop over 4-5 days into mature CTL pre-armed with cytotoxic granules. We now show that TCR activation also triggers Hh signaling during this time which increases levels of Rac1 and thereby promotes centrosome polarization, actin remodelling, granule release and target cell killing. In this way Hh signaling pre-arms CTL with the ability to rapidly polarise the cytoskeleton and deliver the cytotoxic granules within minutes when the CTL encounters a target. Our results reveal molecular parallels between primary cilia and the immunological synapse, highlighting the possible origin of the immunological synapse as a modified cilium.
We would like to thank R. Rohatgi for the MSCV-Ptch1-EYFP construct and the Smo antibodies, S. Munro (Cambridge) for PACT (pericentrin)-RFP, M. Davidson (University of Florida) for Farnesyl-5-TagBFP2, R. Zamoyska for Lck-depleted spleens and D. Fearon, T. Crompton, J. Kaufman, M. A. de la Roche and A. Schuldt for helpful discussions and critical reading of the manuscript and R. Rohatgi and J. Stinchcombe for helpful discussions. We also thank the flow cytometry core facility at CIMR for cell sorting and assistance with the calcium assay. This work was supported by a Wellcome Trust Principal Research Fellowship to GMG (075880) a Wellcome Trust Strategic Award for core facilities at the CIMR (100140) and NIH (R01AR05439, R01GM095941), the Burroughs Wellcome Fund, the David & Lucile F. Packard Foundation, and the Sandler Family Supporting Foundation to JR. The data presented in this paper are provided in the main paper and supplementary materials.