BACKGROUND & AIM: Patients with inflammatory bowel diseases (IBD), specifically those treated with anti-tumor necrosis factor (TNF)a biologics, are at high risk for vaccine-preventable infections. Their ability to mount adequate vaccine responses is unclear. The aim of the study was to assess serologic responses to messenger RNA-Coronavirus Disease 2019 vaccine, and safety profile, in patients with IBD stratified according to therapy, compared with healthy controls (HCs). METHODS: Prospective, controlled, multicenter Israeli study. Subjects enrolled received 2 BNT162b2 (Pfizer/BioNTech) doses. Anti-Gastroenterology 2021;-:1-14 CLINICAL ATspike antibody levels and functional activity, anti-TNFa levels and adverse events (AEs) were detected longitudinally. RE-SULTS: Overall, 258 subjects: 185 IBD (67 treated with anti-TNFa, 118 non-anti-TNFa), and 73 HCs. After the first vaccine dose, all HCs were seropositive, whereas w7% of patients with IBD, regardless of treatment, remained seronegative. After the second dose, all subjects were seropositive, however anti-spike levels were significantly lower in anti-TNFa treated compared with non-anti-TNFa treated patients, and HCs (both P < .001). Neutralizing and inhibitory functions were both lower in anti-TNFa treated compared with non-anti-TNFa treated patients, and HCs (P < .03; P < .0001, respectively). Anti-TNFa drug levels and vaccine responses did not affect anti-spike levels. Infection rate (w2%) and AEs were comparable in all groups. IBD activity was unaffected by BNT162b2. CONCLUSIONS: In this prospective study in patients with IBD stratified according to treatment, all patients mounted serologic response to 2 doses of BNT162b2; however, its magnitude was significantly lower in patients treated with anti-TNFa, regardless of administration timing and drug levels. Vaccine was safe. As vaccine serologic response longevity in this group may be limited, vaccine booster dose should be considered.
T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.
T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion-and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.T cell activation triggers multiple molecular events, including the activation of protein tyrosine kinases (PTKs), formation of multiprotein signaling complexes, and activation of enzymes and transcription factors (25,37,47). Cytoskeletal actin reorganization is also dependent on these events initiated at the T cell-antigen-presenting cell (APC) interface, the immunological synapse (IS). Interference with actin dynamics results in an impaired immune response and can induce T cell anergy (40).We and others (2,4,8,11,12,16) have demonstrated complex molecular events linking T cell antigen receptor (TCR) activation to actin rearrangement. One major pathway, mediated by the activation of multiple PTKs, leads to phosphorylation of the adapter molecules linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). Phosphorylation of SLP76 leads to recruitment of the Nck adapter molecule, which is associated with key regulators of the actin cytoskeleton Wiskott-Aldrich syndrome protein (WASp) and WAVE2.The molecular structure of SLP76 consists of an N-terminal sterile-alpha motif (SAM) (41), an acidic domain containing tyrosine residues subject to phosphorylation, a central prolinerich region, and a C-terminal SH2 domain. Phosphorylation of the tyrosines allows the interaction of SLP76 with the adapter Nck, the Rho-family GEF, VAV, and Itk, all via their SH2 domains (5,8,48,49,51). The interactions of SLP76, Nck, and VAV are essential for the activation of WASp and its recruitment to the IS (51). TCR engagement also induces the association of the SLP76 SH2 domain with the adhesion-and degranulation-promoting adapter protein (ADAP) and to the serine-threonine kinase hematopoietic progenitor kinase 1 (HPK-1) (38). In addition to SLP76, ADAP is capable of binding other proteins, and it is recruited to the IS (26, 35). The role of ADAP in integrin function has been explored (10,23,36,46); however, it...
Natural killer (NK) cells discriminate between healthy cells and virally infected or transformed self-cells by tuning activating and inhibitory signals received through cell surface receptors. Inhibitory receptors inhibit NK cell function by recruiting and activating the tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-1 (SHP-1) to the plasma membrane. However, to date, the guanine nucleotide exchange factor VAV1 is the only direct SHP-1 substrate identified in NK cells. We reveal that the adaptor protein linker for activation of T cells (LAT) as well as phospholipase C-γ1 (PLC-γ1) and PLC-γ2 are SHP-1 substrates. Dephosphorylation of Tyr(132) in LAT by SHP-1 in NK cells abrogated the recruitment of PLC-γ1 and PLC-γ2 to the immunological synapse between the NK cell and a cancer cell target, which reduced NK cell degranulation and target cell killing. Furthermore, the ubiquitylation of LAT by the E3 ubiquitin ligases c-Cbl and Cbl-b, which was induced by LAT phosphorylation, led to the degradation of LAT in response to the engagement of inhibitory receptors on NK cells, which abrogated NK cell cytotoxicity. Knockdown of the Cbl proteins blocked LAT ubiquitylation, which promoted NK cell function. Expression of a ubiquitylation-resistant mutant LAT blocked inhibitory receptor signaling, enabling cells to become activated. Together, these data identify previously uncharacterized SHP-1 substrates and inhibitory mechanisms that determine the response of NK cells.
Protein-protein interactions regulate and control many cellular functions. A multimolecular complex consisting of the adaptor proteins SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kD), Nck, and the guanine nucleotide exchange factor Vav1 is recruited to the T cell side of the interface with an antigen-presenting cell during initial T cell activation. This complex is crucial for regulation of the actin machinery, antigen recognition, and signaling in T cells. We studied the interactions between these proteins as well as the dynamics of their recruitment into a complex that governs cytoskeletal reorganization. We developed a triple-color Förster resonance energy transfer (3FRET) system to observe the dynamics of the formation of this trimolecular signaling complex in live human T cells and to follow the three molecular interactions in parallel. Using the 3FRET system, we demonstrated that dimers of Nck and Vav1 were constitutively formed independently of both T cell activation and the association between SLP-76 and Nck. After T cell receptor stimulation, SLP-76 was phosphorylated, which enabled the binding of Nck. A point mutation in the proline-rich site of Vav1, which abolishes its binding to Nck, impaired actin rearrangement, suggesting that Nck-Vav1 dimers play a critical role in regulation of the actin machinery. We suggest that these findings revise the accepted model of the formation of a complex of SLP-76, Nck, and Vav1 and demonstrate the use of 3FRET as a tool to study signal transduction in live cells.
Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes.
Interaction between the integrin VLA-4 on acute myelogenous leukemia (AML) cells with stromal fibronectin is a decisive factor in chemotherapeutic resistance. In this study, we provide a rationale for a drug repositioning strategy to blunt integrin activation in AML cells and restore their sensitivity to chemotherapy. Specifically, we demonstrate that the nontoxic tellurium compound AS101, currently being evaluated in clinical trials, can abrogate the acquired resistance of AML. Mechanistic investigations revealed that AS101 caused redox inactivation of adjacent thiols in the exofacial domain of VLA-4 after its ligation to stromal fibronectin. This effect triggered cytoskeletal conformational changes that decreased PI3K/Akt/Bcl2 signaling, an obligatory step in chemosensitization by AS101. In a mouse xenograft of AML derived from patient leukemic cells with high VLA-4 expression and activity, we demonstrated that AS101 abrogated drug resistance and prolonged survival in mice receiving chemotherapy. Decreased integrin activity was confirmed on AML cells in vivo. The chemosensitizing activity of AS101 persisted in hosts with defective adaptive and innate immunity, consistent with evidence that integrin deactivation was not mediated by heightening immune attack. Our findings provide a mechanistic rationale to reposition the experimental clinical agent, AS101, to degrade VLA-4-mediated chemoresistance and improve clinical responses in patients with AML. Cancer Res; 74(11); 3092-103. Ó2014 AACR.
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