Dominik Filipp

Regulation of Fyn Through Translocation of Activated Lck into Lipid Rafts

Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinated, and their delivery of function integrated, is unknown. Here we show that lipid rafts function to segregate Lck and Fyn in T cells before activation. Coaggregation of TCR and CD4 leads to Lck activation within seconds outside lipid rafts, followed by its translocation into lipid rafts and the activation of colocalized Fyn. Genetic evidence demonstrates that Fyn activation is strictly dependent on receptor-induced translocation of Lck. These results characterize the interdependence of Lck and Fyn function and establish the spatial and temporal distinctions of their roles in the cellular activation process.

Interaction of the Wiskott–Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells

The Wiskott–Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9ΔPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation.

Enrichment of Lck in Lipid Rafts Regulates Colocalized Fyn Activation and the Initiation of Proximal Signals through TCRαβ

Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCRβ chain. The present study uses a family of murine IL-2-dependent CD4+ T cell clonal variants in which anti-TCRCβ signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag- and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4-associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRCβ responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCRζ:ζ-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4+ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRCβ signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRCβ proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.

Functional Requirements for Signaling through the Stimulatory and Inhibitory Mouse NKR-P1 (CD161) NK Cell Receptors

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56lck. The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.

Lck-dependent Fyn Activation Requires C Terminus-dependent Targeting of Kinase-active Lck to Lipid Rafts

Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active Y505FLck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of Y505FLck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.

The pool of preactivated Lck in the initiation of T-cell signaling: a critical re-evaluation of the Lck standby model.

The initiation of T‐cell receptor (TCR) signaling, based on the cobinding of TCR and CD4‐Lck heterodimer to a peptide–major histocompatibility complex II on antigen presenting cells, represents a classical model of T‐cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine‐based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed ‘standby model of Lck’ posits that resting T‐cells contain an abundant pool of constitutively active Lck (pY394Lck) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394Lck is necessary for the generation of TCR proximal signals in a time‐restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394Lckis likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394Lck‐specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394Lck pool in primary T‐cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.

Gastrointestinal Autoimmunity Associated With Loss of Central Tolerance to Enteric α-Defensins

BACKGROUND & AIMS Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is an autoimmune disorder characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, but patients also develop intestinal disorders. APECED is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE, which regulates immune tolerance) that allow self-reactive T cells to enter the periphery. Enteric α-defensins are antimicrobial peptides secreted by Paneth cells. Patients with APECED frequently have gastrointestinal symptoms and seroreactivity against secretory granules of Paneth cells. We investigated whether enteric α-defensins are autoantigens in humans and mice with AIRE deficiency. METHODS We analyzed clinical data, along with serum and stool samples and available duodenal biopsies from 50 patients with APECED collected from multiple centers in Europe. Samples were assessed for expression of defensins and other molecules by quantitative reverse transcription polymerase chain reaction and flow cytometry; levels of antibodies and other proteins were measured by immunohistochemical and immunoblot analyses. Histologic analyses were performed on biopsy samples. We used Aire(-/-) mice as a model of APECED, and studied the effects of transferring immune cells from these mice to athymic mice. RESULTS Enteric defensins were detected in extraintestinal tissues of patients with APECED, especially in medullary thymic epithelial cells. Some patients with APECED lacked Paneth cells and were seropositive for defensin-specific autoantibodies; the presence of autoantibodies correlated with frequent diarrhea. Aire(-/-) mice developed defensin-specific T cells. Adoptive transfer of these T cells to athymic mice resulted in T-cell infiltration of the gut, loss of Paneth cells, microbial dysbiosis, and the induction of T-helper 17 cell-mediated autoimmune responses resembling those observed in patients with APECED. CONCLUSIONS In patients with APECED, loss of AIRE appears to cause an autoimmune response against enteric defensins and loss of Paneth cells. Aire(-/-) mice developed defensin-specific T cells that cause intestinal defects similar to those observed in patients with APECED. These findings provide a mechanism by which loss of AIRE-mediated immune tolerance leads to intestinal disorders in patients with APECED.

A specific type of membrane microdomains is involved in the maintenance and translocation of kinase active Lck to lipid rafts.

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

Eosinophils from patients with type 1 diabetes mellitus express high level of myeloid alpha-defensins and myeloperoxidase

Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Recently, small cationic α-defensin molecules have been implicated in the pathogenesis of certain inflammatory and autoimmune diseases. The purpose of this study was to assess the α-defensin expression in patients with T1D and elucidate the cellular source of their production. Our results show that 30% of patients exhibit increased levels of α-defensin mRNAs in their capillary blood. Quantitative RT-PCR performed on FACS-sorted granulocytes identified CD15(dull)/CD14(weak) population as the cellular source of α-defensins. Surprisingly, this granulocyte subpopulation displayed augmentation of α-defensin expression in all T1D patients tested. The determination of cell surface markers, expression of cell-specific genes and confocal microscopy identified CD15(dull)/CD14(weak) cells as eosinophils. The presence of transcriptionally active eosinophils in diabetic patients suggests that eosinophils could be a part of an intricate innate immune cellular network involved in the development of diabetes.

TCR Triggering Induces the Formation of Lck–RACK1–Actinin-1 Multiprotein Network Affecting Lck Redistribution

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1–Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR–CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1–Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1–pY394Lck–α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1–Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.