Immune Synapse Formation Research Articles

Primary Human CD4+ T Cells Have Diverse Levels of Membrane Lipid Order That Correlate with Their Function

Membrane lipid microdomains (lipid rafts) play an important role in T cell function by forming areas of high lipid order that facilitate activation. However, their role in regulating T cell differentiation and function remains controversial. In this study, by applying a new approach involving microscopy and flow cytometry, we characterize membrane lipid order in ex vivo primary human CD4(+) T cells. We reveal that differential membrane lipid order dictates the response to TCR stimulation. T cells with high membrane order formed stable immune synapses and proliferated robustly, intermediate order cells had reduced proliferative ability accompanied by unstable immune synapse formation, whereas low order T cells were profoundly unresponsive to TCR activation. We also observed that T cells from patients with autoimmune rheumatic disease had expanded intermediate order populations compared with healthy volunteers. This may be important in dictating the nature of the immune response since most IFN-γ(+)CD4(+) T cells were confined within intermediate membrane order populations, whereas IL-4(+)CD4(+) T cells were contained within the high order populations. Importantly, we were able to alter T cell function by pharmacologically manipulating membrane order. Thus, the results presented from this study identify that ex vivo CD4(+) T cells sustain a gradient of plasma membrane lipid order that influences their function in terms of proliferation and cytokine production. This could represent a new mechanism to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could direct altered immune cell activation in pathology.

Impact of β2 integrin deficiency on mouse natural killer cell development and function

AbstractNatural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

Ab-IL2 fusion proteins mediate NK cell immune synapse formation in epithelial ovarian cancer by polarizing CD25 to the target cell–effector cell interface

Ab-IL2 fusion proteins mediate NK cell immune synapse formation in epithelial ovarian cancer by polarizing CD25 to the target cell–effector cell interface

Proteomic Characterization of Plasma Membrane-proximal T Cell Activation Responses

Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.

Clinical spectrum, pathophysiology and treatment of the Wiskott–Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS), caused by mutations in the WAS gene, is a complex and diverse disorder with X-linked inheritance. This review focuses on recent developments in the understanding of its basic pathophysiology, diverse clinical phenotypes and optimal patient management including novel therapies. The protein encoded by the WAS gene is a multifunctional signaling element expressed in immune and hematopoietic cells that plays a critical role in cytoskeletal reorganization, immune synapse formation and intracellular signaling. The type of specific mutation, its location within the gene and its effect on protein expression play a major role in determining an individual patient's clinical phenotype. Recent clinical observations and molecular studies have created a sophisticated picture of the disease spectrum. The improved outcome of stem cell transplantation from related and unrelated matched donors and promising early results from the first clinical gene therapy trial have added new therapeutic options for these patients. Classic WAS, X-linked thrombocytopenia and X-linked neutropenia are caused by WAS gene mutations, each having a distinct pattern of clinical symptoms and disease severity. New developments in the understanding of these syndromes and novel therapeutic options will have a major impact on the treatment of individuals with WAS mutations.

Special features of quantification of CD8<sup>+</sup>CD38<sup>+ </sup>T-cells by flow cytometry does not represent a good biomarker to monitor the reactivation of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation

CD38 also known as cyclic ADP ribose hydrolase is a surface glycoprotein expressed by several leukocytes, in particular CD4+ and CD8+ T-cells, B-lymphocytes and natural killer cells.(1) CD38 is part of a large family of nucleotide-metabolizing ectoenzymes that also includes CD26, CD39 and CD73. These molecules are clustered in specialized areas of the cell membrane, playing roles as receptor signaling molecules thus regulating cell activation, defense from pathogens, cell migration, immune synapse formation and also life cycle.(2) CD38 is a powerful disease biomarker for several human disorders, including leukemias and myelomas, and also directly involved in the pathogenesis and prognosis of human virus infections. Several potential clinical applications of CD38 as a putative laboratorial biomarker in the diagnosis, prognosis and therapy of viral infections have been previously reported.(2) Elevated CD38 antigen expression on CD8+ T cells has been pointed out as a stronger marker for the risk of chronic HIV disease progression to AIDS.(3) Moreover, recent studies have demonstrated that a parallel decline of CD8+CD38+ lymphocytes and viremia may occur in treated hepatitis B patients, suggesting its role as a biomarker for active infection.(4) It has been proposed that monitoring of the CD8+CD38+ T-cell subset in peripheral blood represents a powerful approach to detect cytomegalovirus (CMV) infections in kidney transplant recipients.(5) More recently, it has been reported that in fact, the monitoring of CD38 high expression in peripheral blood CD8+ lymphocytes is a useful additional diagnostic marker for CMV infection in patients after kidney transplantation, especially when they are at risk of tissue-invasive disease when CMV DNA copies may not be detectable in peripheral blood.(6) The questions surrounding CD38 are still fascinating and continue to prompt novel investigations. One important matter to be clarified is whether the overexpression of the CD38 molecule by circulating T cells may represent a universal biomarker for CMV infection after all modalities of organ transplantation. In order to address this issue, Lino et al.(7) performed a prospective investigation to clarify the applicability of flow cytometric enumeration of CD8+CD38+ T-cells in peripheral blood as a laboratorial indicator of CVM infection/reactivation in allogeneic hematopoietic stem cell (HSCT) recipients. The data presented in this study revealed that, in contrast to those previously published in kidney transplantation, the overexpression of CD38 in circulating CD8+ T cells does not correlate with the presence on CMV antigenemia. The authors present a plausible explanation for this phenomenon, discussing the particularities of CD38 expression during bone marrow repopulation and immune system reconstitution after HSCT. The cyclic nature of CD38 expression during hematopoietic ontogeny may explain the presence of CD38 overexpression even in those HSCT recipients with undetectable antigenemia. It has been reported that CD38 is expressed early in hematopoietic cells, with downregulation during the cell maturation process and re-expression upon cell activation. This knowledge is relevant to aware flow cytometry readers and medical professionals working in the clinical management of transplant recipients that overexpression of CD38 may not be considered a universal biomarker of CMV infection following organ transplantation. The article is well organized, presenting the technical details necessary to ensure its reproducibility, excellent scientific quality and promises a fruitful reading.

Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells

T-cell recognition of peptide–MHC complexes on APCs requires cell–cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5nN after 10s interaction to about 15nN after 30min. In the absence of antigen, or when ICAM-1-negative APC was used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide–MHC complexes.

Lenalidomide Induces Capping of CD20 and Cytoskeleton Proteins to Enhance Rituximab Immune Recognition of Malignant B-Cells

AbstractAbstract 2845Lenalidomide is an immunomodulatory agent that has both direct tumoricidal and immunomodulatory activities which are critical for its clinical activity in the treatment of various hematologic malignancies. This activity is at least in part mediated by enhanced T-cell and NK-cell effector function to eliminate tumor B cells, attributed to restoration of impaired T-cell activity and formation of immunologic synapses. Rituximab is an anti-CD20 monoclonal antibody that is active in the treatment of B-cell lymphomas through a variety of mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Preclinical studies and early clinical trials have shown an enhancement, and potentially synergy, in antitumor activity when lenalidomide is combined with rituximab. In order to further explore the mechanistic basis of this enhancement we investigated the impact of lenalidomide and rituximab on immune synapse formation and ADCC. We have previously shown that the combined use of lenalidomide and rituximab enhances NK cell-mediated immune synapse formation and the resultant cytotoxicity, versus each agent alone. Here we evaluate the molecular events that take place on the cell surface upon exposure of JeKo-1 cells (mantle cell lymphoma) and primary B-CLL cells to lenalidomide alone or lenalidomide plus rituximab. Change in CD20 expression resulting from exposure to vehicle control (0.1% DMSO) or 1 μM lenalidomide for 30 min or 24, 48, 72 hrs was assessed using immunocytochemistry, flow cytometry and isolation of cell membrane-associated proteins followed by Western blotting. At all time points evaluated, levels of cell surface and cell membrane-associated CD20 expression were unchanged in JeKo-1 cells. However, the distribution of CD20 was dramatically altered within 30 minutes after addition of lenalidomide. CD20 redistribution was accompanied by F-actin polymerization and lipid raft aggregation associated with the polarized localization (capping) of a number of proteins including CD20, CD19 and cytoskeleton signaling molecules Rac1 and Vav1, critical regulators of immune synapse formation in effector cells. Of note, other surface proteins involved in signaling such as CD45 were not part of this capping mechanism. By 48 hours of lenalidomide treatment, the majority of JeKo-1 cells (>80%) showed continued capping of CD20. These responses were also seen in primary B-CLL cells, although the effects were variable. In addition, CD20, F-actin and lipid rafts co-localized at the immune synapses formed between JeKo-1 and NK cells treated with either 1 μM lenalidomide for 24 hrs, 0.1% DMSO for 24 hrs followed by 10 μg/ml rituximab for 30 min, or treated sequentially with 1μM lenalidomide for 24 hrs followed by 10 ug/ml rituximab for 30 min. Lenalidomide and rituximab induced similar effects on B-CLL cells and the immune synapses formed between B-CLL and NK cells. We also determined whether formation of lipid rafts and actin cytoskeleton modifications were a prerequisite for CD20 capping. Cholesterol extraction from JeKo-1cells by 5 mM methyl-β-cyclodextrin (MCD) treatment for 30 min led to complete abrogation of lenalidomide-induced capping. The polymerization of the F-actin cytoskeleton and capping of CD20 was also affected, with no impact on cell viability. In addition, MCD treatment inhibited the formation of immunologic synapses between JeKo-1 cells and NK cells treated with 1 μM lenalidomide alone and in cells co-treated with 1 μM lenalidomide and 10 μg/ml rituximab. These data are consistent with a requirement for the integrity of lipid rafts to maintain the capping of CD20 and to potentially mediate lenalidomide enhancement of ADCC by rituximab. Our results further demonstrate that lenalidomide does not down-regulate CD20, but rather induces its polarized localization at the cell surface. The capping of CD20 is accompanied by redistribution of proteins such as Vav1 and Rac1 that become part of the immune synapse complex. Therefore the capping process induced by lenalidomide appears integral to immune synapse formation and may coordinately enhance the clustering of both the CD20 antigen and the attached rituximab, potentially further enhancing its activity, which would support the clinical combination of these agents. Ongoing studies are currently examining the role of the capping process and intracellular signaling cascades in the direct tumoricidal activity of lenalidomide. Disclosures:Gaidarova:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Aukerman:Celgene Corporation: Employment. Daniel:Celgene Corporation: Employment. Chopra:Celgene Corporation: Employment. Lopez-Girona:Celgene Corporation: Employment, Equity Ownership.

Inhibition of Histone Deacetylase 6 Effects on Interaction of Tumor and Immune Cells In Multiple Myeloma

AbstractAbstract 2996Histone deacetylase 6 (HDAC6), a cytoplasmic enzyme, is a member of Class IIb HDAC family and catalyses the deacetylation of histones. Acetylation of histones induces chromatin relaxation leading gene transcription; conversely, deacetylation of histones induces chromatin condensation and silencing of gene transcription. HDAC6 regulates important cellular functions including histone modifications, gene transcription, and viral infection, degradation of misfolded proteins, cell migration, and immune synapse formation. HDAC inhibitors have been developed over the last decade as anti-tumor therapeutic agents in solid tumors and hematologic malignancies including multiple myeloma (MM). Myeloma represents a model system for the development of novel drugs which impact tumor cells as well as accessory cells in the tumor microenvironment. In this study we evaluated the effects of selective HDAC6 inhibition on immune accessory cells using a novel HDAC6 small molecule inhibitor, WT-161 compared with a control HDAC inhibitor trichostatin-A (TsA). Peripheral blood (PBMC) or bone marrow mononuclear cells (BMNC) from healthy donors or MM patients were cultured in the absence or presence of WT-161 (0.25-2.5uM) for 6h to 7 days. We first determined whether WT-161 induces HDAC6 inhibition in normal PBMCs by western blot of acetylated (Lys40)-tubulin, a target for HDAC6 activity. WT-161 induced acetylation of tubulin in PBMCs as early as 6 hours of exposure. Immunophenotypic changes were determined by flow cytometric analysis. Immunophenotypic analysis of PBMCs cultured with WT-161 showed significantly increased expression of PD1 in all immune cell subpopulations including CD4T, CD8T, CD3/CD8/CD56+ NKT and NK cells as well as in CD14+ myeloid cells. Conversely, decreased expression of negative costimulatory molecules CTLA4 and PDL1 was also noted, with no significant change in the expression of positive costimulatory molecules CD28, ICOS and ICOSL. Additionally, culture of PBMCs with WT-161 significantly increased expression of effector/memory T cell marker CXCR3 and decreased expression of CXCR4 in CD4T, CD8T, NKT, NK effector cells as well as CD14+ myeloid cells. WT-161 also induced ICAM1 expression on all immune cell subpopulations. WT-161 effects on the proliferation of immune cells were next assessed by CFSE-flow cytometric analysis. CFSE stained PBMCs were stimulated with anti-CD3 ab and cultured with WT-161 for 7 days. While WT-161 triggered proliferation of CD4T and CD8T cells, proliferation of NK and NKT cells decreased and B cells were unaffected. Interestingly, CD4/CD25/Foxp3 coexpressing regulatory T cells were also significantly diminished in both PBMCs and BMNCs cultured with WT-161 for 4 days. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in the immune effector cells in MM microenvironment. Ongoing analysis of effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures:Raje:AMGEN: Consultancy; Celgene: Consultancy; Novartis: Consultancy; astraZeneca: Research Funding. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson&Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Functional Screening Studies Identify Combinational Activity of PD-L1 and CD200 In Mediating Dysfunctional T Cell Immunological Synapse Formation In Chronic Lymphocytic Leukemia

AbstractAbstract 696The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures:Gribben:Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

A novel mutation of the WAS gene in a patient with Wiskott-Aldrich syndrome presenting with recalcitrant viral warts

Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder characterized by a combination of immunodeficiency, microthrombocytopenia, recalcitrant eczema, and high susceptibility to cancers and autoimmune diseases [1]. WAS has a broad range of clinical phenotypes associated with different mutations in the gene encoding the WAS protein (WASP), which is involved in cell signaling, cell locomotion, and immune synapse formation [2,3].

Sustained LFA‐1 cluster formation in the immune synapse requires the combined activities of L‐plastin and calmodulin

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution - however, not TCR/CD3 relocalization - into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS.

Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

Integrin and CD3/TCR activation are regulated by the scaffold protein AKAP450

During antigen recognition by T cells, membrane receptors and cytoskeletal molecules form a specialized structure at the T cell-antigen-presenting cell junction called the immune synapse (IS). We report a role for the scaffolding protein A-kinase anchoring protein-450 (AKAP450), a member of the A-kinase anchoring protein family, in IS formation and T-cell signaling in antigen- and superantigen-dependent T-cell activation. Suppression of AKAP450 by overexpression of a dominant-negative form or siRNA knockdown disrupted the positioning and conformational activation of lymphocyte function-associated antigen 1 at the IS and impaired associated signaling events, including phosphorylation of phospholipase C-gamma1 and protein kinase C-. AKAP450 was also required for correct activation and phosphorylation of CD3, LAT, and Vav1, key T-cell receptor-activated intracellular signaling molecules. Consistently, antigen-triggered reorientation of the microtubule-organizing center at the IS and interleukin-2 secretion were diminished in AKAP450-disrupted T cells. These results indicate key roles for AKAP450 in the organization and activation of receptor molecules at the IS during T-cell signaling events.

Calpain 4 is not necessary for LFA-1-mediated function in CD4+ T cells.

BackgroundT cell activation and immune synapse formation require the appropriate activation and clustering of the integrin, LFA-1. Previous work has reported that the calpain family of calcium-dependent proteases are important regulators of integrin activation and modulate T cell adhesion and migration. However, these studies have been limited by the use of calpain inhibitors, which have known off-target effects.Methodology/Principal FindingsHere, we used a LoxP/CRE system to specifically deplete calpain 4, a small regulatory calpain subunit required for expression and activity of ubiquitously expressed calpains 1 and 2, in CD4+ T cells. CD4+ and CD8+ T cells developed normally in Capn4F/F:CD4-CRE mice and had severely diminished expression of Calpain 1 and 2, diminished talin proteolysis and impaired casein degradation. Calpain 4-deficient T cells showed no difference in adhesion or migration on the LFA-1 ligand ICAM-1 compared to control T cells. Moreover, there was no impairment in conjugation between Capn4F/F:CD4-CRE T cells and antigen presenting cells, and the conjugates were still capable of polarizing LFA-1, PKC-theta and actin to the immune synapse. Furthermore, T cells from Capn4F/F:CD4-CRE mice showed normal proliferation in response to either anti-CD3/CD28 coated beads or cognate antigen-loaded splenocytes. Finally, there were no differences in the rates of apoptosis following extrinsic and intrinsic apoptotic stimuli.Conclusion/SignificanceOur findings demonstrate that calpain 4 is not necessary for LFA-1-mediated adhesion, conjugation or migration. These results challenge previous reports that implicate a central role for calpains in the regulation of T cell LFA-1 function.

NF‐κ B‐dependent upregulation of ICAM‐1 by HPV16‐E6/E7 facilitates NK cell/target cell interaction

NK cell recognition of tumor cells is mediated by a delicate balance of signals received by MHC class I-binding inhibitory NK cell receptors and activating NK cell receptors, which mainly bind to virus-, stress- or tumor-induced ligands. In addition, adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) and its receptors, the lymphocyte function-associated antigen-1 (LFA-1) and Mac-1, are crucial for immune synapse formation and NK cell-mediated killing. In this study, we show that expression of the adhesion molecule ICAM-1 was rapidly induced by E6 and -E7 oncoproteins of HPV16, -18, -5 and -8, but not of HPV38 and -6 in primary human keratinocytes after retroviral transduction. ICAM-1 was upregulated in E6E7-expressing keratinocytes both at mRNA and protein levels. The observed ICAM-1 upregulation in HPV16-E6E7-expressing keratinocytes was partially dependent on activation of the NF-κB pathway. Importantly, the upregulated ICAM-1 expression in HPV16-E6E7-expressing keratinocytes led to enhanced conjugate formation with NK cells. We previously showed that HPV16-positive cervical carcinomas frequently express low levels of inhibitory NK cell ligands and high levels of activating NK cell ligands. Moreover, levels of the adhesion molecule ICAM-1 are enhanced by HPV16-E6/E7. Therefore, strategies that aim at harnessing NK cells might be beneficial for the treatment of cervical carcinoma.

Quantitative analysis of immune synapse formation using imaging flow cytometry. (130.18)

Abstract Antigen-specific immune responses are initiated via direct cellular contact between an antigen presenting cell (APC) and an effector cell. Early events in the interaction between these two cells involve reorganization of the actin cytoskeleton and recruitment of adhesive and signaling molecules to the immunological synapse (IS), which ultimately results in effector cell activation. Because manual image acquisition and quantitative image analysis are time consuming processes, microscopic analyses of protein recruitment to the IS have remained either qualitative or statistically limited, despite the rich information content inherent in high resolution digital images. Here we describe an objective, statistically robust microscopy-based method for quantifying molecular recruitment to the IS using multispectral imaging flow cytometry, which enables quantitative image analysis of large populations of automatically collected images. The analysis technique uses morphology-based features to identify conjugates, followed by measurement of fluorescence specifically at the conjugate junction. Two models are described, including: 1) actin recruitment to the IS following T cell activation by artificial APC coated with anti-CD3; 2) antigen-specific recruitment of ADAP to the IS within TCR transgenic T cells. These data outline an objective and statistically robust method to rapidly quantify molecular recruitment to the immune synapse using automated high speed microscopy.

Dendritic cells control T cell tonic signaling required for responsiveness to foreign antigen.

Dendritic cells (DCs) are key components of the adaptive immune system contributing to initiation and regulation of T cell responses. T cells continuously scan DCs in lymphoid organs for the presence of foreign antigen. However, little is known about the functional consequences of these frequent T cell-DC interactions without cognate antigen. Here we demonstrate that these contacts in the absence of foreign antigen serve an important function, namely, induction of a basal activation level in T cells required for responsiveness to subsequent encounters with foreign antigens. This basal activation is provided by self-recognition of MHC molecules on DCs. Following DC depletion in mice, T cells became impaired in TCR signaling and immune synapse formation, and consequently were hyporesponsive to antigen. This process was reversible, as T cells quickly recovered when the number of DCs returned to a normal level. The extent of T cell reactivity correlated with the degree of DC depletion in lymphoid organs, suggesting that a full DC compartment guarantees optimal T cell responsiveness. These findings indicate that DCs are specialized cells that not only present foreign antigen, but also promote a "tonic" state in T cells for antigen responsiveness.

WASP: a key immunological multitasker

The Wiskott-Aldrich syndrome protein (WASP) is an important regulator of the actin cytoskeleton that is required for many haematopoietic and immune cell functions, including effective migration, phagocytosis and immune synapse formation. Loss of WASP activity leads to Wiskott-Aldrich syndrome, an X-linked disease that is associated with defects in a broad range of cellular processes, resulting in complex immunodeficiency, autoimmunity and microthrombocytopenia. Intriguingly, gain of function mutations cause a separate disease that is mainly characterized by neutropenia. Here, we describe recent insights into the cellular mechanisms of these two related, but distinct, human diseases and discuss their wider implications for haematopoiesis, immune function and autoimmunity.

Unexpected role of clathrin adaptor AP-1 in MHC-dependent positive selection of T cells.

Trafficking of transmembrane receptors to a specific intracellular compartment is conducted by adaptor molecules that bind to target motifs within the cytoplasmic domains of cargo proteins. We generated mice containing a lymphoid-specific deficiency of AP-1 using RNAi knockdown technology. Inhibition of AP-1 expression in thymocytes blocks progression from double-positive immature thymocytes, resulting in complete absence of CD4(+) single-positive thymocytes and severe reduction of CD3(+)CD8(+) single-positive thymocytes. Analysis of the contribution of AP-1 deficiency on the interaction between mature CD4(+) T cells and antigen-presenting cells revealed that AP-1 is essential to efficient immune synapse formation and associated T cell activation, suggesting a possible mechanism of AP-1 function in thymocyte development.