Cell membranes are structurally heterogeneous, composed of discrete domains with unique physical and biological properties. Membrane domains can form through a number of mechanisms involving ...lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft. How rafts form remains a current topic in membrane biology. We review here evidence of structuring of rafts by the cortical actin cytoskeleton. This includes evidence that the actin cytoskeleton associates with rafts, and that many of the structural and functional properties of rafts require an intact actin cytoskeleton. We discuss the mechanisms of the actin-dependent raft organization, and the properties of the actin cytoskeleton in regulating raft-associated signaling events. We end with a discussion of membrane rafts and the actin cytoskeleton in T cell activation, which function synergistically to initiate the adaptive immune response.
Cell membranes are laterally organized into functionally discrete domains that include the cholesterol-dependent membrane “rafts.” However, how membrane domains are established and maintained remains ...unresolved and controversial but often requires the actin cytoskeleton. In this study, we used fluorescence resonance energy transfer to measure the role of the actin cytoskeleton in the co-clustering of membrane raft-associated fluorescent proteins (FPs) and FPs targeted to the nonraft membrane fraction. By fitting the fluorescence resonance energy transfer data to an isothermal binding equation, we observed a specific co-clustering of raft-associated donor and acceptor probes that was sensitive to latrunculin B (Lat B), which disrupts the actin cytoskeleton. Conversely, treating with jasplakinolide to enhance actin polymerization increased co-clustering of the raft-associated FPs over that of the nonraft probes. We also observed by immunoblotting experiments that the actin-dependent co-clustering coincided with regulation of the raft-associated Src family kinase Lck. Specifically, Lat B decreased the phosphorylation of the C-terminal regulatory tyrosine of Lck (Tyr505), and combining the Lat B with filipin further decreased the Tyr505 phosphorylation. Furthermore, the Lat B-dependent changes in Lck regulation required CD45 because no significant changes occurred in treated T cells lacking CD45 expression. These data define a role for the actin cytoskeleton in promoting co-clustering of raft-associated proteins and show that this property is important toward regulating raft-associated signaling proteins such as Lck.
Pneumococcal diseases remain prevalent despite available polysaccharide and conjugate vaccines. This phase 1/2 study evaluated safety/tolerability and immunogenicity of a novel 24-valent pneumococcal ...vaccine (ASP3772) based on high-affinity complexing of proteins and polysaccharides.
Pneumococcal vaccine–naïve adults aged 18–85 years were randomized to receive either ASP3772 or PCV13 (13-valent conjugate vaccine). Participants received a single intramuscular injection of ASP3772 (1-, 2-, or 5-µg dose per polysaccharide) or PCV13. A separate, nonrandomized group of PCV13-vaccinated participants (65–85 years) received PPSV23 (23-valent polysaccharide vaccine). Assessments were obtained through Day 7 for reactogenicity, through Day 30 for safety and tolerability, and through Month 6 for serious adverse events. Immunogenicity was measured at Day 30 using assays for functional opsonophagocytic activity (OPA) and pneumococcal serotype-specific anticapsular polysaccharide immunoglobulin G for each serotype.
In both age cohorts, the most frequently reported local reactions were self-limited tenderness and pain after ASP3772 at all dose levels or after PCV13, occurring within 2–3 days. Fatigue, headache, and myalgia were the most frequently reported systemic reactions following either vaccine. Robust OPA responses for all serotypes were observed across all ASP3772 dose groups in both age cohorts. Older adults (aged 65–85 years) who received ASP3772 had significantly higher immune responses to several PCV13 serotypes and all non-PCV13 serotypes than participants who received PCV13. OPA responses to the ASP3772 5-µg dose were significantly higher for several serotypes in naïve participants than in older adults with prior exposure to PCV13 who were administered PPSV23 in this study.
These results demonstrate that ASP3772 is well tolerated, highly immunogenic, and in adults may offer significantly broader protection than existing pneumococcal vaccines.
Clinicaltrials.gov: NCT03803202
Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity ...re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells. MGD006 is designed to be rapidly cleared, and therefore requires continuous delivery. In a mouse model of continuous administration, MGD006 eliminated engrafted KG-1a cells (an AML-M0 line) in human PBMC (peripheral blood mononuclear cell)-reconstituted NSG/β2m(-/-) mice at doses as low as 0.5 μg/kg per day for ~7 days. MGD006 binds to human and cynomolgus monkey antigens with similar affinities and redirects T cells from either species to kill CD123-expressing target cells. MGD006 was well tolerated in monkeys continuously infused with 0.1 μg/kg per day escalated weekly to up to 1 μg/kg per day during a 4-week period. Depletion of circulating CD123-positive cells was observed as early as 72 hours after treatment initiation and persisted throughout the infusion period. Cytokine release, observed after the first infusion, was reduced after subsequent administrations, even when the dose was escalated. T cells from animals with prolonged in vivo exposure exhibited unperturbed target cell lysis ex vivo, indicating no exhaustion. A transient decrease in red cell mass was observed, with no neutropenia or thrombocytopenia. These studies support clinical testing of MGD006 in hematological malignancies, including AML.
•Pn-MAPS24v is a novel 24-valent pneumococcal candidate vaccine developed using the MAPS technology•The safety and immunogenicity of three Pn-MAPS24v dose levels were assessed vs PCV13 in toddlers ...primed with PCV13•All tested Pn-MAPS24v dose levels showed acceptable safety and tolerability profiles•Each Pn-MAPS24v dose level elicited IgG and OPA responses for all 13 common and 10 of the 11 unique S. pneumoniae serotypes•These findings support progression of clinical trials of Pn-MAPS24v to the infant population
Pneumococcal conjugate vaccines (PCVs) significantly reduced pneumococcal disease burden. Nevertheless, alternative approaches for controlling more serotypes are needed. Here, the safety, tolerability, and immunogenicity of a 24-valent (1/2/3/4/5/6A/6B/7F/8/9N/9V/10A/11A/12F/14/15B/17F/18C/19A/19F/20B/22F/23F/33F) pneumococcal vaccine based on Multiple Antigen-Presenting System (MAPS) technology (Pn-MAPS24v) was assessed in toddlers.
In this phase 1, blinded, dose-escalation, active-controlled multicenter study conducted in the United States (September/2020–April/2022), 12–15-month-old toddlers primed with three doses of 13-valent PCV (PCV13) were randomized 3:2 to receive a single dose of one of three Pn-MAPS24v dose levels (1 μg/2 μg/5 μg per polysaccharide) or PCV13 intramuscularly. Reactogenicity (within 7 days), treatment-emergent adverse events (TEAEs, within 180 days), serious/medically attended adverse events (SAEs/MAAEs, within 180 days), and immunogenicity (serotype-specific anti-capsular polysaccharide immunoglobulin G IgG and opsonophagocytic activity OPA responses at 30 days post-vaccination) were assessed.
Of 75 toddlers enrolled, 74 completed the study (Pn-MAPS24v 1 μg/2 μg/5 μg: 15/14/16, PCV13: 29). Frequencies of local (60 %/67 %/31 %) and systemic events (67 %/67 %/75 %) in the Pn-MAPS24v 1 μg/2 μg/5 μg and the PCV13 (55 %, 79 %) groups were in similar ranges. TEAEs were reported by 47 %/40 %/63 % of Pn-MAPS24v 1 μg/2 μg/5 μg recipients and 52 % of PCV13 recipients. No vaccine-related SAE was reported. At 30 days post-vaccination, for each of the 13 common serotypes, ≥93 % of participants in each group had IgG concentrations ≥0.35 μg/mL; >92 % had OPA titers ≥lower limit of quantitation (LLOQ), except for serotype 1 (79 %). For 7/11 unique serotypes (2/8/9N/11A/17F/22F/33F), at all dose levels, ≥78 % of Pn-MAPS24v recipients in each group had IgG concentrations ≥0.35 μg/mL and 80 %-100 % had OPA titers ≥LLOQ.
In 12–15-month-old toddlers, a single dose of Pn-MAPS24v showed an acceptable safety profile, regardless of dose level; AEs were reported at similar frequencies by Pn-MAPS24v and PCV13 recipients. Pn-MAPS24v elicited IgG and OPA responses to all common and most unique serotypes. These results support further clinical evaluation in infants.
Background
New treatment options with improved safety and novel mechanisms of actions are needed for patients with peanut allergy.
Objectives
To evaluate the safety, tolerability, and immunogenicity ...of ASP0892, a peanut DNA vaccine, after intradermal (id) or intramuscular (im) administration in adult or adolescent patients with peanut allergy in two phase 1 studies.
Methods
ASP0892 or placebo was administered every 2 weeks for a total of 4 doses. The doses were 1 mg or 4 mg id or 4 mg im for adults, and 1 mg or 4 mg id for adolescents. Immunologic parameters were assessed longitudinally.
Results
Thirty‐one adults (mean age 24.3 years, 17 males) received ASP0892 (9, 8, 8 patients for 1 mg id, 4 mg id or 4 mg im, respectively) or placebo (2 patients/group). Twenty adolescents (mean age 14.2 years, 11 males) received ASP0892 (8 patients/group) or placebo (2 patients/group). In both studies, the most common treatment‐emergent adverse event (TEAE) was injection site pruritus. No deaths or treatment withdrawal were related to TEAEs. No serious TEAEs related to treatment were observed in adult or adolescent patients. ASP0892 treatment led to modest increases in allergen‐specific IgG and/or IgG4 in adults (1 mg id, 4 mg im) and adolescents (1 mg id, 4 mg id). No improvements in clinical outcomes, including double‐blind placebo‐controlled food challenge, were found after ASP0892 treatment.
Conclusions
In two phase 1 studies, ASP0892 was well tolerated with modest but not clinically relevant changes in immune responses.
ClinicalTrials.gov Identifiers
NCT02851277, NCT03755713.
This study evaluates the safety, tolerability, and immunogenicity of a peanut vaccine, ASP0892, after intradermal or intramuscular injection in adults and adolescents with peanut allergy in two phase 1 clinical studies. Both studies of ASP0892 (ARA‐LAMP‐Vax) vaccine showed no significant safety issues and modest but non‐relevant modifications in clinical or immune responses in adults or adolescents with peanut allergy. The safety and immunogenicity potential of ASP0892 may warrant future explorations of LAMP‐Vax vaccines for immunotherapeutic treatment of other diseases. Ara h, Arachis hypogaea; id, intradermal; Ig, immunoglobulin; im, intramuscular; LAMP, lysosomal‐associated membrane protein.Abbreviations: ARA, arachis; id, intradermal; Ig, immunoglobulin; im, intramuscular; LAMP, lysosomal‐associated membrane protein.
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a prevalent phosphoinositide in cell membranes, with important functions in cell signaling and activation. A large fraction of PIP2 associates with the ...detergent-resistant membrane “raft” fraction, but the functional significance of this association remains controversial. To measure the properties of raft and nonraft PIP2 in cell signaling, we targeted the PIP2-specific phosphatase Inp54p to either the raft or nonraft membrane fraction using minimal membrane anchors. Interestingly, we observed that targeting Inp54p to the nonraft fraction resulted in an enrichment of raft-associated PIP2 and striking changes in cell morphology, including a wortmannin-sensitive increase in cell filopodia and cell spreading. In contrast, raft-targeted Inp54p depleted the raft pool of PIP2 and produced smooth T cells void of membrane ruffling and filopodia. Furthermore, raft-targeted Inp54p inhibited capping in T cells stimulated by cross-linking the T cell receptor, but without affecting the T cell receptor-dependent Ca2+ flux. Altogether, these results provide evidence of compartmentalization of PIP2-dependent signaling in cell membranes such as predicted by the membrane raft model.
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Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions.
This study aimed to determine whether inherent ...qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT).
We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT.
Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6−CRTH2+ and CCR6+CRTH2−). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation.
Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.
T Cell Signal Regulation by the Actin Cytoskeleton Chichili, Gurunadh R.; Westmuckett, Andrew D.; Rodgers, William
Journal of biological chemistry/The Journal of biological chemistry,
05/2010, Letnik:
285, Številka:
19
Journal Article
Recenzirano
Odprti dostop
T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which ...activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.
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