Dendritic Cells Actin’ Out

Adaptive immunity in vertebrates typically initiates through the encounter of a specialized antigen-presenting cell (APC) called a dendritic cell (DC) and a naïve CD4+ helper T cell. During this process, these two cells interact closely through an interface known as the immunological synapse (IS). The IS consists of antigens displayed on the surface of the DC on MHC class II molecules, which become recognized by T cell receptors (TCRs) on the helper T cell surface. Additionally, costimulatory molecules and adhesion ligands are also critically involved in the IS. Previous work has demonstrated that adequate IS formation is multifocal, consisting of many contacts between the DC and the T cell. This multifocal synapse is necessary for optimal delivery of TCR and costimulatory signals that activate the naïve CD4+ T cell, a process known as T cell priming. Many previous studies have mainly focused on the T cell components and how these are regulated during IS formation, for example, through active recruitment of TCRs to the surface and sequential endosomal recycling of these molecules once they are engaged. Much less is known about how DCs, the most potent APCs in adaptive immune initiation, regulate components of the multifocal IS to optimize T cell priming.

Actin is a fundamental structural molecule of the cell cytoskeleton that highly accumulates towards the plasma membrane on the DC face of the IS. This polarized recruitment of actin is critical for T cell priming. Actin dynamics in cells are highly complex and at the basic level begin with the polymerization of a monomeric form of actin (G-actin) into a filamentous form (F-actin). On the plasma membrane, this polymerization is initiated through the Arp2/3 complex which becomes activated to enable actin filament formation through different nucleation promoting factor proteins, including the Wiskott-Aldrich-Syndrome protein (WASP) and the WASP-family verprolin-homologous protein (WAVE) regulatory complex (WRC). Clinically, the importance of actin polymerization is underscored by the fact that mutations in WAS, the gene encoding WASP, in humans results in Wiskott-Aldrich Syndrome, a disease which manifests in blood clotting problems, allergic inflammation including eczema, and combined immunodeficiency resulting in recurrent infections. A similar disease has also been observed in patients with mutations in ARPC1B, a gene encoding for a component of the Arp2/3 complex. It may be plausible that inadequate or aberrant DC activation of T cells may underlie some clinical manifestations of diseases involving mutations in proteins involved in actin polymerization. In support of this, WASP-deficiency in DCs is known to prevent DC and T cell contacts and IS stability, which may result in defective adaptive immune responses, including those important for combatting recurrent infections. However, the role for WRC, the other nucleation promoting factor in actin polymerization at plasma membrane, in DC-directed formation of the IS for T cell priming remained unclear.

            Addressing questions of actin dynamics and specifically the role for the WRC in DC patterning of the IS for T cell priming, Leithner and colleagues report in Jounal of Cell Biology important findings in a paper titled “Dendritic cell actin dynamics control contact duration and priming efficiency at the immunological synapse.” The study employs state-of-the-art microscopy and other cell biological and functional assays, to unravel how WRC-mediated actin polymerization empowers DCs with an optimal capacity for T cell priming. The authors begin by confirming the critical role for actin polymerization in DCs by using mycalolide B, a drug that irreversibly severs actin filaments and prevents actin polymerization. Treatment of DCs with this drug causes DCs to lose their F-actin rich plasma membrane veil structures, resulting in these DCs adopting a spherical shape. Importantly, in the presence of antigens and antigen-specific naïve CD4+ T cells, mycalolide B-treated DCs lose their ability to form a multifocal IS with the helper T cells, instead forming a monofocal IS configuration. As a result of DC actin cytoskeleton disruption, naïve CD4+ T cells are impaired in activation and proliferation, confirming the critical role of DC actin dynamics for DC-driven T cell priming

            The authors next sought to address the role for WRC in DC-directed actin dynamics for multifocal IS formation.  The authors generated DCs from hem1-deficient mice, as hem1 is a crucial component of the WRC in cells from the hematopoietic system (such as DCs). Hem1-deficient DCs, lacking a functional WRC, showed reduced actin at the plasma membrane of the DC face of the IS compared to wild type (WT) DCs. Moreover, live-imaging using hem1-deficient DCs from the Lifeact-eGFP reporter revealed a role for the WRC in lateral dynamics of actin foci at the DC synaptic interface, as hem1-deficient DCs showed more stabilized actin foci at the IS. Functionally, hem1-deficient DCs showed defective ability compared to WT DCs in activating antigen-specific CD4+ T cells on a population level. On a single-cell level, the CD4+ T cells activated by hem1-deficient DCs could proliferate as robustly and commit to producing as much interleukin-2, a cytokine whose production early in T cell priming correlates with TCR signaling, as CD4+ T cells activated by WT DCs.  These results suggest that in the absence of the WRC, DCs still retain a capacity of activating T cells, but WRC-deficient DCs activate fewer T cells than WT DCs. How then might the WRC in DCs optimize T cell priming on a population level?

            The authors noticed that hem1-deficient DCs form a significantly larger interaction with antigen-specific T cells compared to WT DCs. Live cell microscopy also revealed that the contact time between antigen-loaded hem1-deficient DCs and antigen-specific CD4+ T cells was considerably higher than with WT DCs. In turn, however, the hem1-deficient DCs contacted fewer antigen-specific CD4+ T cells. The authors also used electron microscopy to observe the interface between WT or hem1-deficient DCs and antigen-specific CD4+ T cells. This revealed that the interface between the synapses in the hem1-deficient DCs and the T cells showed an over-representation of 35-55nm distances corresponding to the size of DC-expressed adhesion ligand ICAM-1 and the T cell-expressed integrin LFA-1.  This suggested that WRC-driven actin dynamics in DCs regulates adhesion to antigen-specific T cells. Surface-expressed ICAM-1 in DCs is known to be immobilized to the actin-cytoskeleton by an activated phosphorylated form of the ezrin-radixin-moesin (ERM) proteins. Indeed, hem1-deficient DCs showed more phosphorylated ERM proteins compared to WT DCs. This suggests that ICAM-1 is more highly immobilized when WRC is absent from DCs, and this potentially could result in higher LFA-1 interaction with the T cells, resulting in stronger adhesion. To address whether the ICAM-1 and LFA-1 interaction were indeed regulated by WRC in DCs, the authors used integrin-deficient antigen-specific T cells and showed that WT and hem1-deficient DCs were equally capable of activating integrin-deficient T cells. Thus, the authors propose a mechanism by which WRC-directed actin dynamics in DCs regulates adhesion between DCs and antigen-specific CD4+ T cells during IS patterning. This mechanism may then allow DCs to engage as many antigen-specific CD4+ T cells as possible during an immune response to maximize T cell priming. 

As most of the experiments so far were done using very well-defined in vitro conditions, the authors then addressed whether WRC-mediated actin dynamics in DCs would have a functional consequence in vivo. Using adoptive transfer system of WT or hem1-deficient DCs and antigen-specific CD4+ T cells in vivo, the authors utilized intravital imaging of mouse lymph nodes and observed significantly higher interaction times between hem1-deficient DCs and antigen-specific CD4+ T cells, recapitulating the in vitro results. Lastly, the authors also found that there were significantly fewer antigen-specific CD4+ T cells interacting with hem1-deficient DCs compared to WT DCs in vivo, although the T cells that interacted with hem1-deficient DCs did not have a defect in proliferation. These results again mirrored the in vitro findings, illustrating that while WRC-driven actin dynamics by DCs may not necessarily impair T cell activation on a single-cell level, WRC in DCs serves to regulate the adhesion to antigen-specific T cells, presumably to maximize the number of antigen-specific CD4+ T cells that DCs encounter and activate during initiation of the immune response. 

Overall, this study reveals that actin dynamics, though critical for formation of the multifocal IS that supports optimal T cell priming, are also actively regulated by DCs through the WRC. The molecular mechanism the authors suggest concerns the role for WRC in mediating lateral actin dynamics which destabilizes phosphorylated ERM proteins, limiting them from immobilizing ICAM-1 and as a result regulating cell adhesion with the antigen-specific T cell through LFA-1. Thus, WRC optimizes DC-T cell contact time during the initiation of an immune response, presumably to maximize the number of T cells that the DC may readily activate. In my opinion, this paper benefits from a richness of experimental approaches and cutting-edge microscopic techniques that cement a role for the WRC in DCs in the dynamicity of the IS that fosters T cell priming. Whether WRC or other actin modifying proteins in DCs play roles in other DC-dependent functions during an immune response remains unknown. For example, DCs also play fundamental roles in directing helper T cell differentiation, for instance by guiding Th1 versus Th2 cell fates. Actin dynamics through WRC may play a role in these responses, as the contact duration and TCR signaling strength between DCs and naïve CD4+ T cells has been proposed to guide T cell differentiation. In any case, this paper shows how a fundamental component of the cell, the actin cytoskeleton, is exploited by DCs to empower them with an ability to prime CD4+ helper T cells. This raises the possibility that actin dynamics in DCs may be main distinguishing features that allows them to be the most potent APCs in initiation of adaptive immunity.

By Carlos Castellanos

Primary study here

Fluorescence microscopy images of synapses formed between mature WT or hem1-deficient DCs and T cells stained with phalloidin (red) and DAPI (blue). Scale bar: 5 µm.