Mutations affecting the actin regulator WD repeat–containing protein 1 lead to aberrant lymphoid immunity

Contribution of homozygous and compound heterozygous missense mutations in VWA2 to Alzheimer’s disease

Regulation of the Src-related tyrosine kinase Lck is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LckY505F is controlled by Hsp90 (M. J. Bijlmakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lck in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lck constructs expressed in COS-7 cells, we show that the presence of activating Lck mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lck. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lck kinase activity or activation-dependent phosphorylation at serines 42 and 59 or tyrosine 394. By binding to the ATP-binding site, the Src family inhibitor PP2 reduced ubiquitination and overcame the need for Hsp90 monitoring of active Lck. We conclude that the levels of active Lck are influenced by two opposing processes, targeting for degradation by ubiquitination and rescue from degradation by Hsp90 monitoring. Based on the PP2 result, we propose that activation-induced conformational changes of the Lck kinase domain instigate both regulatory processes.

Use of synthetic short interfering RNAs (siRNAs) to study gene function has been limited by an inability to selectively analyze subsets of cells in complex populations, low and variable transfection efficiencies, and semiquantitative assays for measuring protein down-regulation. Intracellular flow cytometry can overcome these limitations by analyzing populations at the single-cell level in a high-throughput and quantitative fashion. Individual cells displaying a knockdown phenotype can be selectively interrogated for functional responses using multiparameter analysis. Lck-specific siRNA was delivered into Jurkat T cells or peripheral blood mononuclear cells (PBMCs) to suppress endogenous Lck expression. Transfected cells were fluorescently stained for intracellular Lck and analyzed using multiparameter flow cytometry. The Lck(lo) Jurkat subpopulation was selectively analyzed for CD69 up-regulation and phospho-states of signaling proteins following T-cell receptor (TCR) stimulation. Surface expression levels of CD4 and CD8 on transfected CD3+ gated PBMCs were correlated with intracellular Lck levels. A subpopulation of Jurkat cells with reduced levels of Lck was clearly resolved from cells with wildtype levels of Lck. Both CD69 up-regulation and ZAP70 phosphorylation were suppressed in Lck(lo) cells when compared with those in Lck(hi) cells upon TCR stimulation. Knockdown of intracellular Lck in primary T lymphocytes reduced surface expression of CD4 in a dose-dependent manner. Multiparameter flow cytometry is a powerful technique for the quantitative analysis of siRNA-mediated protein knockdown in complex hard-to-transfect cell populations.

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

BackgroundInherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the...

CRISPR/Cas9 Mediated Gene Repair in Inherited Bleeding Disorders

Signalling by innate immune cells is critical to shaping the adaptive immune response to microbial infection. In this issue of The EMBO Journal, Labzin etal reveal that the adaptive immune system can instruct the innate response to adenovirus infection. In human macrophages, antibody-coated adenovirus triggers a novel TRIM21-dependent pathway that activates the NLRP3 inflammasome and the secretion of IL-1β.

Aprominent pathology textbook used in the United States includes an image illustrating the renal histopathology caused by malignant hypertension. The legend describes striking “onion skin” changes of a renal arteriole. Curiously, a sea of mononuclear inflammatory cells surrounding this arteriole is overlooked both in the legend and in the related text. Moreover, nothing regarding inflammation or immune reactions is discussed. This lack of attention to inflammatory cells is, however, not surprising. Although many experimental studies have implicated inflammation in hypertension, these have largely been performed in experimental animals; there is no proof that inflammation contributes to human hypertension. In fact, some anti-inflammatory or immune-suppressing drugs (eg, nonsteroidal anti-inflammatory drugs and cyclosporine) paradoxically cause hypertension in humans, likely via off-target effects. Often the term “inflammation” is used in the context of cardiovascular disease as a catchall referring to nonspecific phenomena, such as elevation of C-reactive protein or the presence of macrophages in a tissue. Most clinicians and investigators find this vague and difficult to understand. Even more puzzling is that many studies now implicate the adaptive immune response, and in particular, lymphocytes, in hypertension and vascular disease. Traditionally, bacterial, viral, or tumor antigens activate this arm of immune defense. As such, it has been hard to imagine how adaptive immunity could be involved in a disease such as hypertension. In this article, we will attempt to address some of these puzzling questions. We will briefly review components of the innate and adaptive immune response, discuss data from many groups, including our own, that suggest that common forms of hypertension are immune mediated, and provide a working hypothesis of how signals from the central nervous system trigger an immune response that causes hypertension. ### General Concepts Regarding Inflammation and Immunity #### Innate Immunity The first line of defense against pathogens is the innate immune response. Important components of this system include epithelial …

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.

Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context-which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.

Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse.

Integrins CD49a (alpha1), CD49b (alpha2) and CD103 (alphaE) are highly expressed on tumor infiltrating lymphocytes (TILs) compared to circulating lymphocytes. Interactions of the integrins with known ligands support retention of lymphocytes in peripheral tissues; thus, they may be considered retention integrins (RI). T cells in peripheral tissues expressing RI have a cytotoxic phenotype and RI+ TIL are associated with improved patient survival. Despite their importance in retention of tumor reactive TILs, little is known about pathways inducing RI expression, except that TGFβ induces CD103. We hypothesized that other cytokines, TLR agonists, and T cell receptor (TCR) stimulation can induce RI expression by circulating T cells. We stimulated PBMC with cytokines or TLR agonists after TCR stimulation, and evaluated RI expression by flow cytometry after 2-7 days. Both CD103 and CD49a are induced by TGFβ, most strikingly on activated T cells. IL2 and IL10 also influence CD49a expression. CD49b expression is increased by IL2, IL4 and IL10, although this depends on T cell subset and activation state. TLR2, TLR6 and TLR7 agonists also appear to induce CD49a and CD49b. In conclusion, our results support our hypothesis by showing that RI expression is tightly regulated by cytokine and TCR stimulation. Furthermore these regulating factors have different effects among the RI, suggesting a different role for each.

Defense from infection or cancer is provided by coordinated actions of the innate and adaptive immune system, including B lymphocytes that generate antibodies (Abs) against pathogens or tumor antigens. B cells establish immune synapses with antigen-presenting cells during activation to initiate Ab-production, which may include entrance into a germinal center (GC) reaction to generate very high-affinity Abs. Establishment of an immune synapse requires polarity proteins to coordinate uptake and processing of external antigens. For this, there is a highly conserved family of partitioning defective (Par) proteins that mediates cell polarity in multiple cell types. In particular, Par-3, a member of the Par polarity complex (Par-3, aPKC, Par-6), is a key protein that localizes to the immune synapse and is required for B cell polarization and immune synapse formation. Recently, we showed that LKB1, the mammalian homolog of Par-4 and a regulator of energy metabolism, controls B cell activation, GC formation and differentiation into Ab-producing plasma cells. However, roles for LKB1 in B cell polarity, immune synapse formation and anti-tumor immunity are unknown. Based on previous studies showing that LKB1 induces cell polarity and co-localizes with Par-3, we hypothesize that LKB1 is required for Par complex assembly and controls immune synapse formation during B cell activation. Subcellular analysis of LKB1 by immunofluorescence microscopy in mouse primary B cells reveals LKB1 co-localization with aPKC in membrane protrusions required for immune synapse formation. Interestingly, B cell specific LKB1 knock out (BKO) mice exhibit mislocalization of aPKC/Par complex, loss of lymphoid tissue polarity and spontaneous B cell activation that results in the formation of giant GCs and secretion of proinflammatory cytokines and chemokines. Similar inflammatory profiles with T cell chemoattractants are known to recruit CD8+ cytotoxic T lymphocytes (CTLs) into solid tumors, such as melanoma. These similarities suggest that LKB1 inactivated B cells in the tumor microenvironment could stimulate T cell infiltrates into solid tumors. Our studies suggest that physiologic inactivation of LKB1 in B cells controls B cell activation, GC formation and potentially infiltration of T cells in the tumor microenvironment during anti-tumor responses. We are currently utilizing an exciting genetic mouse melanoma model to examine the role of LKB1 in promoting anti-tumor immunity. Together, our studies assess LKB1 as a target for augmenting adaptive immunity against cancer. Citation Format: Laura Jimenez, Lynnea Waters, Diane N.H. Kim, Nicole Walsh, Michael A. Teitell. Roles for LKB1 at the immune synapse during B-cell activation and antitumor responses [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B46.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen presenting cells (APC)1. T cell signaling is initiated at these contacts when surface-expressed antigen receptors (TCR) recognize peptide fragments (antigens) of pathogens bound to Major Histocompatibility Complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse (IS)3, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft2. T cell recognition of pMHC and the adhesion ligand Intercellular Adhesion Molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse (IS)4–5, which is characterized by central accumulation of TCR5, adjacent to a secretory domain3, both surrounded by an adhesive ring4–5. Although accumulation of TCR at the IS center correlates with T cell function4, this domain is itself largely devoid of TCR signaling activity5–6, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic IS periphery4–5. Here we show that centrally accumulated TCR is located on the surface of extracellular microvesicles that bud at the IS center. Tumor susceptibility gene 101 (TSG101)6 sorts TCR for inclusion in microvesicles, while vacuolar protein sorting 4 (VPS4) 7–8 mediates scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APC.

RasGRP links T-cell receptor signaling to Ras