CLIP-170 is essential for MTOC repositioning during T cell activation by regulating dynein localisation on the cell surface.

The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging. Inhibition of CLIP-170 phosphorylation impaired both MTOC repositioning and interleukin-2 (IL-2) expression. T cell stimulation induced some fraction of dynein to colocalise with CLIP-170 and undergo plus-end tracking. Concurrently, it increased dynein in minus-end-directed movement. It also increased dynein relocation to the centre of the contact surface. Dynein not colocalised with CLIP-170 showed both an immobile state and minus-end-directed movement at a velocity in good agreement with the velocity of MTOC repositioning, which suggests that dynein at the immunological synapse may pull the microtubules and the MTOC. Although CLIP-170 is phosphorylated by AMP-activated protein kinase (AMPK) irrespective of stimulation, phosphorylated CLIP-170 is essential for dynein recruitment to plus-end tracking and for dynein relocation. This indicates that dynein relocation results from coexistence of plus-end- and minus-end-directed translocation. In conclusion, CLIP-170 plays an indispensable role in MTOC repositioning and full activation of T cells by regulating dynein localisation.

VASP Regulates NK Cell Lytic Granule Convergence.

NK cells eliminate viral-infected and malignant cells through a highly orchestrated series of cytoskeletal rearrangements, resulting in the release of cytolytic granule contents toward the target cell. Central to this process is the convergence of cytolytic granules to a common point, the microtubule-organizing center (MTOC), before delivery to the synapse. In this study, we show that vasodialator-stimulated phosphoprotein (VASP), an actin regulatory protein, localizes to the cytolytic synapse, but surprisingly, shows no impact on conjugate formation or synaptic actin accumulation despite being required for human NK cell-mediated killing. Interestingly, we also find that a pool of VASP copurifies with lytic granules and localizes with lytic granules at the MTOC. Significantly, depletion of VASP decreased lytic granule convergence without impacting MTOC polarization. Using the KHYG-1 cell line in which lytic granules are in a constitutively converged state, we find that either VASP depletion or F-actin destabilization promoted spreading of formerly converged granules. Our results demonstrate a novel requirement for VASP and actin polymerization in maintaining lytic granule convergence during NK cell-mediated killing.

Polarity of CD4+ T cells towards the antigen presenting cell is regulated by the Lck adapter TSAd.

Polarization of T cells towards the antigen presenting cell (APC) is critically important for appropriate activation and differentiation of the naïve T cell. Here we used imaging flow cytometry (IFC) and show that the activation induced Lck and Itk adapter T cell specific adapter protein (TSAd), encoded by SH2D2A, modulates polarization of T cells towards the APC. Upon exposure to APC presenting the cognate antigen Id, Sh2d2a-/- CD4+ T cells expressing Id-specific transgenic T cell receptor (TCR), displayed impaired polarization of F-actin and TCR to the immunological synapse (IS). Sh2d2a-/- T-cells that did polarize F-actin and TCR still displayed impaired polarization of PKCξ, PAR3 and the microtubule-organizing center (MTOC). In vitro differentiation of activated Sh2d2a-/- T cells was skewed towards an effector memory (Tem) rather than a central memory (Tcm) phenotype. A similar trend was observed for Id-specific TCR Sh2d2a-/- T cells stimulated with APC and cognate antigen. Taken together our data suggest that TSAd modulates differentiation of experienced T cells possibly through polarization of CD4+ T cells towards the APC.

The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling.

By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.

Analysis of Microtubules and Microtubule-Organizing Center at the Immune Synapse.

The immune synapse (IS) is a specialized structure that enables cell-cell communication between immune cells. As such, it involves direct cell-to-cell contact. It is sustained by cytoskeletal components that allow the intracellular polarization of different organelles and the surface re-organization of signaling and adhesion receptors. The tubulin-based cytoskeleton is a key player in IS formation and signaling. We describe methods to analyze through Western blot and microscopy analysis the polarization to the IS of the centrosome, also known as microtubule-organizing center (MTOC), the dynamics of microtubule growth and polymerization from the MTOC to the IS and the activation of signaling molecules.

Measurement of Lytic Granule Convergence After Formation of an NK Cell Immunological Synapse.

Natural killer (NK) cells contain specialized lysosome-related organelles termed lytic granules allowing them to mediate cytotoxicity against tumorigenic or virally infected target cells. NK cells polarize their lytic granules toward a target cell via the microtubule-organizing center (MTOC). Prior to that, however, lytic granules converge to the MTOC along microtubules utilizing minus-end-directed microtubule motors. Herein we describe how to visualize and quantify lytic granule convergence using confocal microscopy to gain quantitative insight into NK cell cytotoxicity and its regulation.

Studying the T Cell-Astrocyte Immune Synapse.

In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus).

Significant Differences in Antigen-Induced Transendothelial Migration of Human CD8 and CD4 T Effector Memory Cells.

OBJECTIVE: Circulating human T effector memory cell (TEM) recognition of nonself MHC (major histocompatibility complex) molecules on allograft endothelial cells can initiate graft rejection despite elimination of professional antigen-presenting cells necessary for naive T-cell activation. Our previous studies of CD4 TEM have established that engagement of the T-cell receptor not only activates T cells but also triggers transendothelial migration (TEM) by a process that is distinct from that induced by activating chemokine receptors on T cells, being slower, requiring microtubule-organizing center-directed cytolytic granule polarization to and release from the leading edge of the T cell, and requiring engagement of proteins of the endothelial cell lateral border recycling compartment. Although CD4 TEM may contribute to acute allograft rejection, the primary effectors are alloreactive CD8 TEM. Whether and how T-cell receptor engagement affects TEM of human CD8 TEM is unknown. APPROACH AND RESULTS: We modeled TEM of CD8 TEM across cultured human microvascular endothelial cells engineered to present superantigen under conditions of venular shear stress in vitro in a flow chamber. Here, we report that T-cell receptor engagement can also induce TEM of this population that similarly differs from chemokine receptor-driven TEM with regard to kinetics, morphological manifestations, and microtubule-organizing center dynamics as with CD4 TEM. However, CD8 TEM do not require either cytolytic granule release or interactions with proteins of the lateral border recycling compartment. CONCLUSIONS: These results imply that therapeutic strategies designed to inhibit T-cell receptor-driven recruitment based on targeting granule release or components of the lateral border recycling compartment will not affect CD8 TEM and are unlikely to block acute rejection in the clinic.

HkRP3 is a microtubule-binding protein regulating lytic granule clustering and NK cell killing.

NK cells provide host defense by killing viral-infected and cancerous cells through the secretion of preformed lytic granules. Polarization of the lytic granules toward the target cell is dependent on an intact microtubule (MT) network as well as MT motors. We have recently shown that DOCK8, a gene mutated in a primary immunodeficiency syndrome, is involved in NK cell killing in part through its effects on MT organizing center (MTOC) polarization. In this study, we identified Hook-related protein 3 (HkRP3) as a novel DOCK8- and MT-binding protein. We further show that HkRP3 is present in lytic granule fractions and interacts with the dynein motor complex and MTs. Significantly, depletion of HkPR3 impaired NK cell cytotoxicity, which could be attributed to a defect in not only MTOC polarity, but also impaired clustering of lytic granules around the MTOC. Our results demonstrate an important role for HkRP3 in regulating the clustering of lytic granules and MTOC repositioning during the development of NK cell-mediated killing.

The newly found functions of MTOC in immunological response.

The MTOCs are present in all eukaryotic cells. In animal somatic cells, the MTOC function is played by a centrosome, which contains centrioles and PCM. The traditional view is that the MTOC is responsible for the organization of microtubular structures (the intracellular network, cilia, and flagella) in interphase cells, and the formation of the mitotic and meiotic spindle apparatus which is required for the partitioning of chromosomes in dividing cells. Recent evidence suggests that MTOC also plays a key role in the engagement of molecular motors, directional transport of granules, and polarization of subcellular structures and molecules. All of these functions are crucial for targeted cytotoxicity and the regulation of immune cells. In this review, we focus on the ultrastructural and molecular aspects of MTOCs in various aspects of immune cell functions, with specific emphasis on the formation of the IS and targeted cell killing.

Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity.

Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC.

Rapid activation receptor- or IL-2-induced lytic granule convergence in human natural killer cells requires Src, but not downstream signaling.

Natural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase-dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation.

The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.

Anti-microtubule organizing center with microtubule by autoimmune target test is also useful serological marker in rheumatoid arthritis evaluation.

Seropositivity of rheumatoid factor (RF) or anti-cyclic citrullinated peptide antibody (anti-CCP) is one domain of scoring system in new American College of Rheumatology/European League against Rheumatism classification criteria for rheumatoid arthritis (RA). We investigated usefulness of antiperinuclear factor (APF) and autoantibody to microtubule organizing center with microtubule (anti-MTOC-MT), which was detected by autoimmune target (AIT) test, as serological markers for the diagnosis of early RA. The test results of 3,503 patients from the outpatient clinic of The Hospital for Rheumatic Diseases who underwent test for RF, APF, anti-CCP and anti-MTOC-MT simultaneously for the work-up of RA were analyzed. Four kinds of tests showed same results only in 53.1% (1,861/3,503) of all subjects. The kappa coefficient between each test was distributed from -0.011 to 0.622. The agreement was best between RF and anti-CCP (kappa coefficient: 0.622), but the agreement was poor between anti-MTOC-MT and other 3 tests (kappa coefficient: 0.007 to -0.025). In both of RF- and anti-CCP-negative patients, the patients who showed positive result for APF were 4.6% (160/3,503) and for anti-MTOC-MT were 13.2% (464/3,503). RF and anti-CCP positivity could not include all of APF and anti-MTOC-MT positivity. Anti-MTOC-MT detected by AIT test was independent serological marker, and it might be also helpful for the diagnosis of early RA. Therefore, the combined detection of all four serologic markers can be useful for the evaluation of suspected RA patients.

Cytokine secretion by CD4+ T cells at the immunological synapse requires Cdc42-dependent local actin remodeling but not microtubule organizing center polarity.

Cytokine secretion by T lymphocytes plays a central role in mounting adaptive immune responses. However, little is known about how newly synthesized cytokines, once produced, are routed within T cells and about the mechanisms involved in regulating their secretions. In this study, we investigated the role of cytoskeleton remodeling at the immunological synapse (IS) in cytokine secretion. We show that a key regulator of cytoskeleton remodeling, the Rho GTPase Cdc42, controls IFN-γ secretion by primary human CD4+ T lymphocytes. Surprisingly, microtubule organizing center polarity at the IS, which does not depend on Cdc42, is not required for cytokine secretion by T lymphocytes, whereas microtubule polymerization is required. In contrast, actin remodeling at the IS, which depends on Cdc42, controls the formation of the polymerized actin ring at the IS, the dynamic concentration of IFN-γ-containing vesicles inside this ring, and the secretion of these vesicles. These results reveal a previously unidentified role of Cdc42-dependent actin remodeling in cytokine exocytosis at the IS.

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells.

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.

GSK3-mediated instability of tubulin polymers is responsible for the failure of immature CD4+CD8+ thymocytes to polarize their MTOC in response to TCR stimulation.

Although mature T cells divide and differentiate when they receive strong TCR stimulation, most immature CD4+CD8+ thymocytes die. The molecular basis for this marked difference in response is not known. Observations that TCR-stimulated CD4+CD8+ thymocytes fail to polarize their microtubule-organizing center (MTOC), one of the first events that occurs upon antigen activation of mature T cells, suggests that TCR signaling routes in immature and mature T cells diverge early and upstream of MTOC polarization. To better understand the source of the divergence, we examined the molecular basis for the difference in TCR-mediated MTOC polarization. We show that unstable microtubules are a feature of immature murine CD4+CD8+ thymocytes, which also exhibit higher levels of glycogen synthase kinase 3 (GSK3) activity, a known inhibitor of microtubule stability. Importantly, CD4+CD8+ thymocytes gained the ability to polarize their MTOC in response to TCR signals when GSK3 activity was inhibited. GSK3 inhibition also abrogated TCR-mediated apoptosis of immature thymocytes. Together, our results suggest that a developmentally regulated difference in GSK3 activity has a major influence on immature CD4+CD8+ thymocyte versus mature T-cell responses to TCR stimulation.

Cytoskeletal stabilization of inhibitory interactions in immunologic synapses of mature human dendritic cells with natural killer cells.

Human mature dendritic cells (DCs) can efficiently stimulate natural killer (NK)-cell responses without being targeted by their cytotoxicity. To understand this important regulatory crosstalk, we characterized the development of the immunologic synapse between mature DCs and resting NK cells. Conjugates between these 2 innate leukocyte populations formed rapidly, persisted for prolonged time periods and matured with DC-derived f-actin polymerization at the synapse. Polarization of IL-12 and IL-12R to the synapse coincided with f-actin polymerization, while other activating and inhibitory molecules were enriched at the interface between DCs and NK cells earlier. Functional assays revealed that inhibition of f-actin polymerization in mature synapses led to an increase of IFN-γ secretion and cytotoxicity by NK cells. This elevated NK-cell reactivity resulted from decreased inhibitory signaling in the absence of MHC class I polarization at the interface, which was observed on inhibition of f-actin polymerization in DCs. Thus, inhibitory signaling is stabilized by f-actin at the synapse between mature DCs and resting NK cells.

High throughput method to quantify anterior-posterior polarity of T-cells and epithelial cells.

The virologic synapse (VS), which is formed between a virus-infected and uninfected cell, plays a central role in the transmission of certain viruses, such as HIV and HTLV-1. During VS formation, HTLV-1-infected T-cells polarize cellular and viral proteins toward the uninfected T-cell. This polarization resembles anterior-posterior cell polarity induced by immunological synapse (IS) formation, which is more extensively characterized than VS formation and occurs when a T-cell interacts with an antigen-presenting cell. One measure of cell polarity induced by both IS or VS formation is the repositioning of the microtubule organizing center (MTOC) relative to the contact point with the interacting cell. Here we describe an automated, high throughput system to score repositioning of the MTOC and thereby cell polarity establishment. The method rapidly and accurately calculates the angle between the MTOC and the IS for thousands of cells. We also show that the system can be adapted to score anterior-posterior polarity establishment of epithelial cells. This general approach represents a significant advancement over manual cell polarity scoring, which is subject to experimenter bias and requires more time and effort to evaluate large numbers of cells.