title: Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.
abstract: B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.
authors.affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH.
authors.forename: Gregory Lee
authors.initials: GL
authors.lastname: Szeto
authors.affiliation: Department of Biological Engineering, MIT.
authors.forename: Debra
authors.initials: D
authors.lastname: Van Egeren
authors.affiliation: Department of Biology, MIT.
authors.forename: Hermoon
authors.initials: H
authors.lastname: Worku
authors.affiliation: 1] David. H. Koch Institute for Integrative Cancer Research, MIT [2] Department of Chemical Engineering, MIT [3] The Ragon Institute of Harvard, MIT, and MGH.
authors.forename: Armon
authors.initials: A
authors.lastname: Sharei
authors.affiliation: Department of Chemical Engineering, MIT.
authors.forename: Brian
authors.initials: B
authors.lastname: Alejandro
authors.affiliation: Department of Biological Engineering, MIT.
authors.forename: Clara
authors.initials: C
authors.lastname: Park
authors.affiliation: The Ragon Institute of Harvard, MIT, and MGH.
authors.forename: Kirubel
authors.initials: K
authors.lastname: Frew
authors.affiliation: Department of Materials Science &Engineering, MIT.
authors.forename: Mavis
authors.initials: M
authors.lastname: Brefo
authors.affiliation: Department of Chemical Engineering, MIT.
authors.forename: Shirley
authors.initials: S
authors.lastname: Mao
authors.affiliation: David. H. Koch Institute for Integrative Cancer Research, MIT.
authors.forename: Megan
authors.initials: M
authors.lastname: Heimann
authors.affiliation: 1] David. H. Koch Institute for Integrative Cancer Research, MIT [2] Department of Chemical Engineering, MIT.
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.affiliation: Department of Chemical Engineering, MIT.
authors.forename: Klavs
authors.initials: K
authors.lastname: Jensen
authors.affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH [5] Howard Hughes Medical Institute.
authors.forename: Darrell J
authors.initials: DJ
authors.lastname: Irvine
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D019013:CD40 Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D013314:Stress, Mechanical; D014612:Vaccines / Q000276:immunology*
chemical_list: D000941:Antigens; D051940:B7-2 Antigen; D019013:CD40 Antigens; D016207:Cytokines; D014612:Vaccines; D007371:Interferon-gamma