Since the identification of talin as an immune synapse component (17
), there has been considerable interest in understanding its role in LFA-1 function and T cell activation. Using mice with a specific depletion of talin1 in T cells, we now demonstrate for the first time that talin is critical for maintenance of T cell:APC contacts, contact-mediated T cell proliferation, and polarization of stable F-actin to the immune synapse. We found no evidence of defects in TCR signaling contributing to these phenotypes since protein phosphorylation following TCR crosslinking was unaltered and phospho-ZAP70 and PKC-θ localized to the immune synapse of talin1-deficient T cells. Additionally, using live imaging, we observed accumulation of GFP-PH-AKT at the site of T cell:APC contact in the absence of talin1-dependent T cell arrest. Based on our findings, we propose that the current model of LFA-1 activation in T cells should be revised to indicate that talin is dispensable for LFA-1 polarization to the synapse but is required to polarize stabilized F-actin and mediate full T cell arrest.
The defects that we observed in talin1-deficient T cell lymph node homing, APC interactions and proliferation are similar to those previously reported for CD4+ T cells isolated from LFA-1 knockout mice (12
). These shared phenotypes suggest that the defects observed in Talin1Loxp/Loxp
:CD4-Cre T cells may primarily be due to defects in LFA-1 activation. The capacity of LFA-1 to bind its ligand, ICAM-1, is regulated by changes in affinity and clustering following TCR signaling (13
). We have previously shown that talin modulates both components of LFA-1 function (20
). Here, we show that talin1-deficient T cells fail to adhere to ICAM-1 and APCs but retain the ability to cluster LFA-1 at T cell:APC contacts.
Despite having this ability to cluster LFA-1, talin1-deficient T cells failed to fully polarize F-actin to the immune synapse. These findings differed from previous reports suggesting that talin is required for LFA-1 clustering, but not F-actin polarization, during Jurkat superantigen-mediated conjugation (20
). These differences are likely due to the types of stimulation and cell types used: Jurkat T cells treated with superantigen versus antigen-induced primary T cell conjugation. Superantigen-mediated conjugation has previously been shown to bypass proximal TCR signaling (23
), and Jurkat T cell signaling is different from primary T cells (22
). The use of talin knockout mice and antigen-specific primary T cells represents an advance towards better understanding the role of talin1 in T cell adhesion and activation.
While there are clear defects in LFA-1 function in the absence of talin, we found that LFA-1 is still clustered at the immune synapse in talin1-deficient T cells. In other integrin-mediated adhesions, talin1, RapL and kindlins are thought to work in concert to regulate integrin activity (19
). While RapL is not required for T cell adhesion to ICAM-1 (46
), both talin1 and kindlin-III have been independently shown to be important for LFA-1 dependent adhesion of T cells (16
). In this work, we show that RapL and the associated integrin regulatory protein RIAM are localized to the site of T cell:APC contact in talin1-deficient T cells, suggesting that RapL and/or RIAM may potentially be sufficient to cluster LFA-1. However, despite the clustering of LFA-1 at the immune synapse, LFA-1 is not functioning normally in talin1-deficient T cells since the cells have impaired adhesion to both APCs and ICAM-1-coated plates. Together, these findings indicate that although talin1 is dispensable for LFA-1 polarization, it is necessary for LFA-1-mediated adhesive function.
Based on previous studies, it was surprising to find impaired F-actin polarization at the site of T cell:APC contacts in talin1-deficient T cells. Previous work has reported that F-actin polarization to the immune synapse is required for T cell:APC interactions and full T cell proliferation since disruption of F-actin with inhibitors following conjugate formation impairs T cell activation (47
). To better understand the types of F-actin found at the immune synapse, we used two probes of F-actin: Lifeact, which binds to all F-actin present in the cell, and the CH domain of Utrophin, which binds specifically to stable F-actin. Since Utrophin-CH binds to stable F-actin populations, localization of Lifeact alone corresponds to areas of dynamic F-actin (44
). To our knowledge, these probes have not previously been used to characterize the dynamics of F-actin at T cell:APC contact sites.
Live imaging of F-actin dynamics using Lifeact-RUBY and UtrCH-GFP showed that stable F-actin, specifically, failed to polarize to the T cell:APC contact site in talin1-deficient cells. We found that migrating talin1-deficient T cells maintained UtrCH-GFP, a marker of stabilized F-actin, at the uropod without any evidence of its re-distribution following transient T cell:APC contacts, whereas Lifeact-RUBY was enriched at T cell:APC contact sites during transient contacts. This combined with the observation that Arp2/3 and WAVE2 are at the immune synapse in talin1-deficient T cells suggests that actin polymerization at the immune synapse is intact in talin1-deficient cells but that talin is required for F-actin stabilization.
This failure to polarize F-actin in talin1-deficient cells may be due to a requirement for talin to directly bind actin at the immune synapse and provide the link to polarized integrins or it may be due to an inability to recruit vinculin to the immune synapse in the absence of talin1. Supporting the latter hypothesis, we showed that there was a defect in vinculin polarization to the immune synapse in talin1-deficient T cells even though vinculin can bind Arp2/3, which polarized normally to the synapse in the absence of talin1. Therefore, we think it is most likely that talin and vinculin work together to stabilize F-actin at the immune synapse through their interactions with LFA-1 and actin. Indeed, vinculin bound to talin has been shown to play a key role in linking F-actin to focal adhesions(49
). The importance of stabilizing F-actin at the immune synapse is highlighted by the recent findings that T cells lacking leukocyte L-plastin, an actin bundling protein that mediates filamentous actin accumulation at the immune synapse, are not efficiently activated (50
One area of controversy is the relative importance of forming stable T cell:APC interactions for T cell activation. While some in vitro studies show that T cell arrest is required for T cell proliferation and activation (2
), others show that transient interactions are sufficient for T cell activation (6
). In this paper, we demonstrate that talin1 is necessary for T cell arrest and formation of stable interactions between T cells and APCs. The failure of talin1-deficient T cells to arrest and develop stable contacts with APCs in combination with the findings of impaired contact-dependent proliferation supports the hypothesis that T cell arrest is required for T cell proliferation and that talin is a critical regulator of this process. We suspect that talin mediates a connection between F-actin and LFA-1 that is required for complete arrest of T cells on APCs since simply promoting cell-cell contact is not sufficient to restore T cell proliferation in talin1-deficient T cells.
In vivo studies investigating the importance of T cell:APC interactions indicate that T cells form stable, long-lasting interactions with APCs during priming conditions, which contribute to proliferation (10
). However, the requirement of these interactions for T cell proliferation have been challenged with the finding that CD8+ T cells fail to form stable interactions with ICAM-1-deficient APCs in vivo but are still able to proliferate (11
). While we show that talin is required for in vivo CD4+ T cell proliferation in response to ovalbumin, the question of T cell arrest during in vivo priming is hard to address with this system since we cannot determine whether impaired proliferation in talin1-deficient T cells is due to impaired T cell:APC interactions or due to impaired T cell trafficking to lymph nodes. However, our work supports the idea that talin-mediated T cell arrest is required for full CD4+ T cell proliferation and activation.
Here, we provide evidence that talin1 is required for the formation of sustained T cell:APC interactions and T cell contact-dependent proliferation. In addition to its role in regulating LFA-1 function in T cells, it also plays a critical role in maintaining F-actin polarization to the immune synapse. While talin-dependent T cell:APC interactions seem critical in generating a number of T cell:APC responses, future challenges will be to address how talin may support the differentiation of T cells following antigenic challenge and how this differentiation may affect outcomes in response to infection and autoimmunity in vivo.