Isolation and Activation of DC Subsets

DC subsets for vaccination were isolated from spleens and bone marrow of humanized non-tumor-bearing littermates treated with Flt3L as previously described.22 (link) Briefly, DCs were enriched by negative selection from bone marrow cells by incubation with anti-mouse Ter119 (TER-119), anti-human CD14 (RMO52, Beckman Coulter), anti-human CD19 (J3-119, Beckman Coulter), anti-human CD3 (OKT3, BioXCell), anti-human CD34 (My10, BD), and anti-mouse CD45 (30-F11, BD) followed by removal of antibody-bearing cells using sheep anti-rat IgG Dynabeads (Invitrogen). Enriched cells were stained with anti-human CD45-APC-Cy7, anti-HLA-DR-PECy7 (L243), anti-CD123-PerCP-Cy5.5 (6H6), anti-CD141-APC (M80), anti-CD1c-PE (L161), anti-CD11c-PE-CF594, and anti-CD3/CD19/CD20-Pacific Blue. DC subsets were sorted as CD3/CD14/CD19/CD20^–HLA-DR⁺CD11c⁺CD141⁺ (cDC1) and CD3/CD14/CD19/CD20^–HLA-DR⁺CD11c⁺CD141^– (cDC2) using a MoFlo Astrios cell sorter (Beckman Coulter). DCs were activated for 2 hours with 10 µg/mL PolyIC prior to intratumoral injection of 20-25 x 10³ DCs per mouse.

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Lee Y.S., O'Brien L.J., Walpole C.M., Pearson F.E., Leal-Rojas I.M., Masterman K.A., Atkinson V., Barbour A, & Radford K.J. (2021). Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model. Journal for Immunotherapy of Cancer, 9(3), e001963.

Publication 2021

anti-human CD3 (OKT3, anti-mouse CD45 (30-F11, sheep anti-rat IgG Dynabeads MoFlo Astrios cell sorter

Anti cd3 Anti igg Antibody Bone marrow cells Bone marrow tumor Cd123 Cd141 Cd1c Cdc2 Cell Cy5 5 Hla dr Human Mouse Sheep Vaccination

Corresponding Organization : University of Queensland

Other organizations : Princess Alexandra Hospital

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Variable analysis

independent variables

Flt3L treatment of humanized non-tumor-bearing littermates

dependent variables

DC subset isolation from spleens and bone marrow
DC subset enrichment by negative selection
DC subset sorting as cDC1 and cDC2
DC subset activation with PolyIC
Intratumoral injection of 20-25 x 10^3 DCs per mouse

control variables

Humanized non-tumor-bearing littermates

positive controls

None specified

negative controls

None specified

Annotations

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