Recruiting ESCRT to single-chain heterotrimer peptide-MHCI releases antigen-presenting vesicles that stimulate T cells selectively

Immune cells naturally secrete extracellular antigen-presenting vesicles (APVs) displaying peptide:MHC complexes to facilitate the initiation, expansion, maintenance, or silencing of immune responses. Previous work has sought to manufacture and purify these vesicles for cell-free immunotherapies. In this study, APV assembly and release is achieved in non-immune cells by transfecting HEK293T or Expi293F cells with a single-chain heterotrimer (SCT) peptide/major histocompatibility complex I (pMHCI) construct containing an ESCRT- and ALIX-binding region (EABR) sequence appended to the cytoplasmic tail; this EABR sequence recruits ESCRT proteins to induce the budding of APVs displaying SCT pMHCI. A comparison of multiple pMHCI constructs shows that inducing the release of APVs by the addition of an EABR sequence generalizes across SCT pMHCI constructs. Purified pMHCI/EABR APVs selectively stimulate IFN-{gamma} release from T cells presenting their cognate T cell receptor, demonstrating the potential use of these vesicles as a form of cell-free immunotherapy. Significance StatementImmune cells are known to naturally release pMHC-displaying extracellular vesicles (EVs), called antigen-presenting vesicles (APVs), which can orchestrate immune responses either directly or with the aid of antigen-presenting cells (APCs). For decades, researchers have pursued ways to replicate these APVs for immunotherapy by using chemically modified nanoparticles or by engineering the increased expression of APVs from immune cells which are typically low yield. Here we presents a broadly applicable platform for generating high concentrations of pMHCI-displaying APVs that can selectively modulate T cells, demonstrating a significant advance in the engineering of APVs for cell-free immunotherapy. The APVs presented here, and related APVs, could be translated into clinical therapies for modulating cancer progression or regulating autoimmunity in addition to their use as a tool to help characterize how endogenous extracellular vesicles influence the immune system.