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1.  Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse 
The Journal of Experimental Medicine  2013;210(12):2721-2737.
In T cells, PI3K activation in the periphery of the immune synapse leads to PIP3 accumulation that promotes actin polymerization in a pathway important for cytotoxic function.
The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.
PMCID: PMC3832928  PMID: 24190432
2.  NK cell tolerance of self-specific activating receptor KIR2DS1 in individuals with cognate HLA-C2 ligand 
NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC-class I antigens, and protect healthy cells from NK cell-mediated auto-aggression. However, certain activating receptors, including the human killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC-class I. This raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, there was no significant difference in frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2 had significantly reduced frequency of anti-HLA-C2 reactive clones as compared to all other donors. 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly non-cytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present post-transplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.
PMCID: PMC3643210  PMID: 23554313
3.  The Variable Hinge Region of Novel PKCs Determines Localization to Distinct Regions of the Immunological Synapse 
PLoS ONE  2014;9(4):e95531.
The immunological synapse (IS) formed between a T cell and its cognate antigen-presenting cell (APC) enables the directional secretion of cytolytic and inflammatory molecules. Synaptic architecture is established in part by a two-step cascade of novel protein kinase C (nPKC) isozymes. PKCε and PKCη arrive at the IS first, and occupy the entire synaptic membrane. Then, PKCθ accumulates in a smaller zone at the center of the contact. We investigated the molecular basis for this differential recruitment behavior using chimeric nPKC constructs and total internal reflection fluorescence microscopy. Our studies revealed that the V3 linker just N-terminal to the kinase domain plays a crucial role in specifying nPKC localization. Substitution of this linker switched the scope and the kinetics of PKCθ accumulation to that of PKCε and PKCη, and vice versa. Although the V3 was necessary for synaptic compartmentalization, it was not sufficient, as the tandem C1 domains were also required to mediate membrane association. Together, these results suggest a model whereby the V3 linker controls nPKC sub-compartmentalization after initial C1 domain-mediated accumulation at the IS.
PMCID: PMC3994095  PMID: 24751783
4.  IL-2–dependent tuning of NK cell sensitivity for target cells is controlled by regulatory T cells 
The Journal of Experimental Medicine  2013;210(6):1167-1178.
IL-2–dependent adaptive-innate lymphocyte cross talk tunes NK cell reactivity and is limited by T reg cells.
The emergence of the adaptive immune system took a toll in the form of pathologies mediated by self-reactive cells. Regulatory T cells (T reg cells) exert a critical brake on responses of T and B lymphocytes to self- and foreign antigens. Here, we asked whether T reg cells are required to restrain NK cells, the third lymphocyte lineage, whose features combine innate and adaptive immune cell properties. Although depletion of T reg cells led to systemic fatal autoimmunity, NK cell tolerance and reactivity to strong activating self- and non-self–ligands remained largely intact. In contrast, missing-self responses were increased in the absence of T reg cells as the result of heightened IL-2 availability. We found that IL-2 rapidly boosted the capacity of NK cells to productively engage target cells and enabled NK cell responses to weak stimulation. Our results suggest that IL-2–dependent adaptive-innate lymphocyte cross talk tunes NK cell reactivity and that T reg cells restrain NK cell cytotoxicity by limiting the availability of IL-2.
PMCID: PMC3674692  PMID: 23650441
5.  Novel Foxo1–dependent transcriptional programs control Treg cell function 
Nature  2012;491(7425):554-559.
Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses1–4. Foxp3 operates as a late-acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6–10. However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulatorof Treg cell function. Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with Treg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of Treg cells. Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.
PMCID: PMC3771531  PMID: 23135404
6.  Inhibitory Receptor Signaling Destabilizes Immunological Synapse Formation in Primary NK Cells 
Upon engagement of their cognate class I major histocompatibility complex ligands, receptors containing immunotyrosine-based inhibitory motifs (ITIMs) transduce signals that block cytolytic and inflammatory responses. In this manner, ITIM-coupled receptors play a crucial role in maintaining natural killer (NK) cell tolerance toward normal, healthy tissue. A number of studies, mostly using immortalized NK cell lines, have demonstrated that ITIM signaling functions by disrupting the cytolytic immunological synapse formed between an NK cell and its target. However, more recent imaging experiments using primary NK cells have suggested that inhibitory receptor engagement does not antagonize contact formation, casting doubt on the hypothesis that ITIM signals destabilize the synapse. To resolve this issue, we analyzed primary NK cell activation and contact formation on supported lipid bilayers containing controlled combinations of activating and inhibitory ligands. Under these conditions, we observed that ITIM signaling clearly inhibited adhesion, cell arrest, and calcium influx, three hallmarks of synapse formation. These results are consistent with previous reports showing that inhibitory receptors deliver a “reverse stop” signal, and confirm that ITIM signaling functions at least in part by destabilizing cytolytic synapse formation.
PMCID: PMC3841721  PMID: 24348477
NK cell; signal transduction; immunological synapses; ITIM; imaging
7.  Lymphocyte polarity, the immunological synapse and the scope of biological analogy 
Bioarchitecture  2011;1(4):180-185.
Lymphocytes such as T cells, B cells and natural killer (NK) cells form specialized contacts, called immunological synapses, with other cells in order to engage in specific intercellular communication and killing. Synapse formation is associated with the polarization of the microtubule-organizing center (MTOC) toward the contact site, which enables the directional secretion of cytokines and lytic factors. Although MTOC reorientation to the synapse is crucial for lymphocyte function, it has been difficult to study because of technical constraints. We have developed a photoactivation and imaging strategy that enables high-resolution analysis of cytoskeletal dynamics in individual T cells. Using this approach, we have demonstrated that the lipid second messenger diacylglycerol plays a crucial role in promoting MTOC reorientation by recruiting three members of the protein kinase C family to the synapse. Here, I will discuss these results along with studies from other labs, which have explored the role of polarity-inducing protein complexes after synapse formation. I will also propose a two-step model for MTOC reorientation in lymphocytes that reflects what we now know about the subject. Finally, I will consider the extent to which lymphocyte polarity resembles analogous cell polarity systems in other cell types.
PMCID: PMC3210514  PMID: 22069511
polarity; T cell; microtubule; cytoskeleton; signaling; lymphocyte; chemical biology
8.  A Cascade of Protein Kinase C Isozymes Promotes Cytoskeletal Polarization in T Cells 
Nature immunology  2011;12(7):647-654.
Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell is driven by the accumulation of diacylglycerol at the immunological synapse (IS). The mechanisms that couple diacylglycerol to the MTOC are not known. Using single-cell photoactivation of the T cell receptor, we demonstrated that three distinct protein kinase C (PKC) isoforms are recruited by diacylglycerol to the IS in two steps. PKC-ε and PKC-η accumulated first in a broad region of membrane, while PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation, and that PKC-ε and PKC-η operated redundantly to promote PKC-θ recruitment and subsequent polarization responses. These results establish a previously uncharacterized role for PKCs in T cell polarity.
PMCID: PMC3119370  PMID: 21602810
9.  Microtubule-organizing center polarity and the immunological synapse: protein kinase C and beyond 
Cytoskeletal polarization is crucial for many aspects of immune function, ranging from neutrophil migration to the sampling of gut flora by intestinal dendritic cells. It also plays a key role during lymphocyte cell–cell interactions, the most conspicuous of which is perhaps the immunological synapse (IS) formed between a T cell and an antigen-presenting cell (APC). IS formation is associated with the reorientation of the T cell’s microtubule-organizing center (MTOC) to a position just beneath the cell–cell interface. This cytoskeletal remodeling event aligns secretory organelles inside the T cell with the IS, enabling the directional release of cytokines and cytolytic factors toward the APC. MTOC polarization is therefore crucial for maintaining the specificity of a T cell’s secretory and cytotoxic responses. It has been known for some time that T cell receptor (TCR) stimulation activates the MTOC polarization response. It has been difficult, however, to identify the machinery that couples early TCR signaling to cytoskeletal remodeling. Over the past few years, considerable progress has been made in this area. This review will present an overview of recent advances, touching on both the mechanisms that drive MTOC polarization and the effector responses that require it. Particular attention will be paid to both novel and atypical members of the protein kinase C family, which are now known to play important roles in both the establishment and the maintenance of the polarized state.
PMCID: PMC3459186  PMID: 23060874
cell polarity; cytoskeleton; lymphocyte; protein kinase C; signal transduction; T cell
10.  Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells 
The Journal of Cell Biology  2011;192(4):675-690.
Signaling from immunotyrosine-based inhibitory motifs (ITIMs) neutralizes activating signals by inducing a retraction of NK cells from the surface of stimulatory cells.
Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell–activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.
PMCID: PMC3044118  PMID: 21339333
11.  Shouts, whispers and the kiss of death: directional secretion in T cells 
Nature immunology  2008;9(10):1105-1111.
T cells use secreted soluble factors for highly specific intercellular communication and targeted cell killing. This specificity is achieved first through T cell receptor–mediated recognition of complexes of peptide and major histocompatibility complex displayed by appropriate antigen-presenting cells and then by the directed secretion of cytokines and lytic factors into the immunological synapse between the T cell and antigen-presenting cell. Studies have begun to probe the molecular basis for this synaptic secretion and have also shown that T cells release chemokines and certain inflammatory factors through a multidirectional pathway directed away from the synapse. Thus, the mode of secretion seems to be tailored to the intended function of the secreted molecule.
PMCID: PMC2905669  PMID: 18800163

Results 1-11 (11)