IL-15 <i>Trans</i>-Presentation Is an Autonomous, Antigen-Independent Process

Kenesei, Ádám; Volkó, Julianna; Szalóki, Nikoletta; Mocsár, Gábor; Jambrovics, Károly; Balajthy, Zoltán; Bodnár, Andrea; Tóth, Katalin; Waldmann, Thomas A.; Vámosi, György

doi:10.4049/jimmunol.2100277

Cited by 8 publications

(12 citation statements)

References 64 publications

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“…SEE contains two distinct binding sites for MHC II and one on the T cell receptor, thereby crosslinking these proteins [ 31 ]. Previously we used this model system to study Kv1.3 enrichment at the IS and IL-15 trans-presentation [ 25 , 32 ]. Here, we found that WT and NON-CON Kv1.3 variants were dominantly localized in the plasma membrane ( Figure 3 A,B), whereas the ΔC mutant exhibited a higher intracellular fraction ( Figure 3 C).…”

Section: Resultsmentioning

confidence: 99%

“…We used Jurkat CD4 + T cell lines stably expressing different forms of Kv1.3 channels tagged with mGFP at the N-terminus: wild-type (WT), a ΔC mutant (the last 84 amino acids including the PDZ-binding domain deleted), and NON-CON mutant (point mutation in the poreregion (W384F), abolishing conductance but preserving motion of the gating charges). For control experiments, IL-2Rα was stably transfected into Jurkat cells expressing WT mGFP-Kv1.3 by retroviral transduction, as described in [ 32 ]. We used Raji B cells as antigen presenting cells that can form synapses with Jurkat cells.…”

Section: Methodsmentioning

confidence: 99%

“…Enrichment of Kv1.3 at the IS was calculated as described [ 32 ]. Briefly, the ratio of the average pixel intensity inside the IS and in the whole cell membrane was calculated from confocal images by using a MATLAB script as follows:

where I IS is the intensity of a pixel in the IS, N IS is the number of such pixels, I tot is the intensity of a pixel anywhere in the cell membrane (including the IS), and N tot is the number of all pixels.…”

Section: Methodsmentioning

confidence: 99%

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Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Sebestyén

Nagy

Mocsár

et al. 2022

IJMS

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Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Sebestyén

Nagy

Mocsár

et al. 2022

IJMS

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“…interacting with F-actin are at the periphery of the IS (pSMAC) 59 , although variations occur depending on the T cell type 60,61 . In a similar process, IL-15 transpresentation, an APC expressing the IL-15Rα/IL-15 complex interacts with a T cell expressing the IL-2/15Rβ and γc subunits of the receptor inducing survival signals 22,29 . The molecules participating in these intercellular proteinprotein interactions get enriched at the IS; MHC II and IL-15Rα move jointly to the IS when either ligand (antigen or IL-15) is present 22 .…”

Section: Discussionmentioning

confidence: 99%

Molecular-scale complexes of IL-2/15 receptors and MHC molecules persist but become more mobile, while microdomain-level aggregates are dispersed on actin-free membrane blebs of T cells

Vámosi,

Mocsár,

Vereb

et al. 2024

Preprint

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Earlier we found that interleukin-2 and -15 receptors (IL-2R, IL-15R) and MHC I and II, all proteins essential in T cell activation, form co-clusters at different hierarchic levels in lipid rafts of T cells. To investigate whether the membrane-associated actin cytoskeleton has a role in organizing these clusters, we used membrane blebs formed spontaneously on starved human T lymphoma cells for comparison, as they have a smooth surface without cytoskeletal connections. STED microscopy showed that the patchy cell surface distribution of IL-2Rα, IL-15Rα, MHC I and the lipid raft marker GM1 ganglioside observed on intact cell membranes became more homogenous on blebs. Spatial autocorrelation analysis revealed that cluster sizes diminished. Fluorescence correlation spectroscopy and single molecule tracking showed a two- to four-fold increase in the mobility of the investigated proteins. FRET efficiencies between homo- or hetero-associated proteins remained constant or decreased moderately and the associated fractions of protein pairs determined by fluorescence cross-correlation spectroscopy remained unchanged or increased. Our results show that the higher-order organization of the membrane becomes more homogeneous, clusters become smaller and the mobility of molecules increases on actin-free membrane blebs, but the investigated protein-protein interactions are preserved and do not depend on the actin cytoskeleton.

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“…Using two humanized immunocompetent orthotopic bladder tumor models and a clinically-relevant investigational approach, including use of the fully-human, Fc-optimized anti-CD40 agonist antibody 2141-V11 that is under active clinical evaluation for the treatment of bladder cancer and other indications (NCT05126472, NCT04059588, NCT04547777), we noted substantial improvement in the therapeutic activity of 2141-V11-induced CD40 agonism through concurrent stimulation with exogenous IL-15, including robust generation of both primary and systemic memory responses. This combination therapy approach was found to critically depend on an intact cDC1-CD8 T cell axis, and is likely to be leveraging the observed upregulation (but incomplete occupancy) of surface IL-15Rα, which has been shown to be capable of incorporating exogenous IL-15 into the trans-presentation pathway (42, 43). Other groups have also observed the ability of CD40 agonists to overcome SOCS3-mediated impairment of CD4 T cell help induced by immunotherapy with common cytokine receptor gamma chain (γc) family members like IL-15 (44), providing additional mechanistic support for the combined therapeutic activation of the CD40 and IL-15 pathways.…”

Section: Discussionmentioning

confidence: 99%

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

Wong¹,

Smith²,

Angulo-Lozano³

et al. 2023

Preprint

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CD40 is a central co-stimulatory receptor implicated in the development of productive anti-tumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway have demonstrated dose limiting toxicities with minimal clinical activity to date, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe an important role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of trans-presented IL-15/IL-15Ra surface complexes, particularly by cross-presenting cDC1s, and associated enrichment of activated CD8 T cells within the bladder tumor microenvironment. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, however, they lack high levels of IL-15Ra at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Combination therapy enhances the crosstalk between Batf3-dependent cDC1s and CD8 T cells, driving robust primary anti-tumor activity and further stimulating long-term systemic anti-tumor memory responses associated with circulating memory-phenotype T and NK cell populations. Collectively, these data reveal an important role for IL-15 in mediating anti-tumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to evaluate combinations of these promising therapeutics for the treatment of patients with bladder cancer.

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IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process

Abstract: Positive and Negative Roles of the Trans-Acting T Cell Factor-1 for the Acquisition of Distinct Ly-49 MHC Class I Receptors by NK Cells

Cited by 8 publications

References 64 publications

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Molecular-scale complexes of IL-2/15 receptors and MHC molecules persist but become more mobile, while microdomain-level aggregates are dispersed on actin-free membrane blebs of T cells

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

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