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Screening self-peptides for recognition by mouse alloreactive CD8⁺ T cells using direct ex vivo multimer staining.

ABSTRACT: Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring recognition of the various epitopes by these alloreactive T cells. For complete details on the use and execution of this protocol, please refer to Son et al. (2021).¹.

SUBMITTER: Son ET

PROVIDER: S-EPMC9792548 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Screening self-peptides for recognition by mouse alloreactive CD8<sup>+</sup> T cells using direct ex vivo multimer staining.

Son Eric T ET Paul-Heng Moumita M Leong Mario M Wang Chuanmin C Hill Alexandra E AE Denkova Martina M Purcell Anthony W AW Mifsud Nicole A NA Sharland Alexandra F AF

STAR protocols 20221215 1

Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring re ...[more]

PMID: 36525346

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Project description:Selectively depleting the pathogenic T cells is a fundamental strategy for the treatment of allograft rejection and autoimmune disease since it retains the overall immune function of host. The concept of killer artificial antigen-presenting cells (KaAPCs) has been developed by co-coupling peptide-major histocompatibility complex (pMHC) multimer and anti-Fas monoclonal antibody (mAb) onto the polymeric microparticles (MPs) to induce the apoptosis of antigen-specific T cells. But little information is available about its in vivo therapeutic potential and mechanism. In this study, polyethylenimine (PEI)-coated poly lactic-co-glycolic acid microparticle (PLGA MP) was fabricated as a cell-sized scaffold to covalently co-couple H-2Kb-Ig dimer and anti-Fas mAb for the generation of alloantigen-presenting and apoptosis-inducing MPs. Intravenous infusions of the biodegradable KaAPCs prolonged the alloskin graft survival for 43 days in a single MHC-mismatched murine model, depleted the most of H-2Kb-alloreactive CD8+ T cells in peripheral blood, spleen, and alloskin graft in an antigen-specific manner and anti-Fas-dependent fashion. The cell-sized KaAPCs circulated throughout vasculature into liver, kidney, spleen, lymph nodes, lung, and heart, but few ones into local allograft at early stage, with a retention time up to 36 h in vivo. They colocalized with CD8+ T cells in secondary lymphoid organs while few ones contacted with CD4+ T cells, B cells, macrophage, and dendritic cells, or internalized by phagocytes. Importantly, the KaAPC treatment did not significantly impair the native T cell repertoire or non-pathogenic immune cells, did not obviously suppress the overall immune function of host, and did not lead to visible organ toxicity. Our results strongly document the high potential of PLGA MP-based KaAPCs as a novel antigen-specific immunotherapy for allograft rejection and autoimmune disorder. The in vivo mechanism of alloinhibition, tissue distribution, and biosafety were also initially characterized, which will facilitate its translational studies from bench to bedside.

Dataset Information

Screening self-peptides for recognition by mouse alloreactive CD8⁺ T cells using direct ex vivo multimer staining.

Publications

Screening self-peptides for recognition by mouse alloreactive CD8<sup>+</sup> T cells using direct ex vivo multimer staining.

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Dataset Information

Screening self-peptides for recognition by mouse alloreactive CD8+ T cells using direct ex vivo multimer staining.

Publications

Screening self-peptides for recognition by mouse alloreactive CD8<sup>+</sup> T cells using direct ex vivo multimer staining.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Screening self-peptides for recognition by mouse alloreactive CD8⁺ T cells using direct ex vivo multimer staining.