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4-1BB costimulation promotes CAR T cell survival through noncanonical NF-?B signaling.

ABSTRACT: Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19⁺ hematologic malignancies. 4-1BB-costimulated CAR (BB?) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28?) T cells. 4-1BB signaling improves T cell persistence even in the context of 28? CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. To specifically study signal transduction by CARs, we developed a cell-free, ligand-based activation and ex vivo culture system for CD19-specific CAR T cells. We observed greater ex vivo survival and subsequent expansion of BB? CAR T cells when compared to 28? CAR T cells. We showed that only BB? CARs activated noncanonical nuclear factor ?B (ncNF-?B) signaling in T cells basally and that the anti-CD19 BB? CAR further enhanced ncNF-?B signaling after ligand engagement. Reducing ncNF-?B signaling reduced the expansion and survival of anti-CD19 BB? T cells and was associated with a substantial increase in the abundance of the most pro-apoptotic isoforms of Bim. Although our findings do not exclude the importance of other signaling differences between BB? and 28? CARs, they demonstrate the necessary and nonredundant role of ncNF-?B signaling in promoting the survival of BB? CAR T cells, which likely underlies the engraftment persistence observed with this CAR design.

SUBMITTER: Philipson BI

PROVIDER: S-EPMC7883633 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.

Philipson Benjamin I BI O'Connor Roddy S RS May Michael J MJ June Carl H CH Albelda Steven M SM Milone Michael C MC

Science signaling 20200331 625

Clinical response to chimeric antigen receptor (CAR) T cell therapy is correlated with CAR T cell persistence, especially for CAR T cells that target CD19<sup>+</sup> hematologic malignancies. 4-1BB-costimulated CAR (BBζ) T cells exhibit longer persistence after adoptive transfer than do CD28-costimulated CAR (28ζ) T cells. 4-1BB signaling improves T cell persistence even in the context of 28ζ CAR activation, which indicates distinct prosurvival signals mediated by the 4-1BB cytoplasmic domain. ...[more]

PMID: 32234960

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Project description:Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.

Dataset Information

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-?B signaling.

Publications

4-1BB costimulation promotes CAR T cell survival through noncanonical NF-κB signaling.

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