B cells loading DRibbles antigen could re-activate effector T cells.

<p>(A) The schematic diagram outlines the experiment protocol. C57/BL6 mice or BALB/c mice were intro-node vaccinated with DRibbles respectively. Lymphocytes were collected from lymph nodes (LN) of vaccinated C57/BL6 mice or BALB/c mice on day 7 after immunization. B cells purified from wide type C57/BL6 mice were stimulated with DRibbles for 6 hours and then washed 3 times with PBS. The lymphocytes were co-incubated with DRibbles or DRibbles-loaded B cells or B cells alone for 72 hours (n = 5). (B) The supernatants were harvested for detection of IFN-γ by ELISA. (C) CD8⁺ T cells were purified from the vaccinated C57/BL6 mice, and then co-incubated with B cells plus DRibbles (with DCs plus DRibbles at 1∶1 ratio or B cell alone as control) at the indicated ratio for 72 hours. IFN-γ in the supernatants was tested via ELISA. (D and E) CD4⁺ T cells were purified from the vaccinated C57/BL6 mice, and then co-incubated with B cells plus DRibbles at the indicated ratio for 72 hours. IFN-γ (D) and IL-4 (E) in the supernatants were tested via ELISA. (F) CD8⁺ T cells purified from the DRibbles-vaccinated C57/BL6 mice were co-incubated at 1∶1 ratio with B cells (from wild type, TLR4-, TLR2- or MyD88-deficient mice) plus DRibbles for 72 hours. IFN-γ in the supernatants was tested via ELISA. (G) CD8⁺ T cells were purified from the Hep1-6-DRibbles vaccinated C57/BL6 mice, and then co-incubated at 1∶1 ratio with B cells plus Hep1-6-, B16F10- or BNL-DRibbles for 72 hours respectively. IFN-γ in the supernatants was tested via ELISA. Data which are presented were obtained as a result of triplicates.</p

DRibbles induced proliferation and activation of B cells in vitro.

<p>(A) Purified B cells were labeled with CFSE and then co-incubated with DRibbles (DRs), whole tumor cell lysate (Lys) or LPS for 5 days. The proliferation of B cells was accessed by flow cytometry. (B) Purified B cells were co-incubated with DRibbles for 3 days and collected for staining with the indicated Abs or isotype-matched control Abs (gray filled area). The expression of H2-K^b, I-A^b, CD86 and CD40 on B cells was analyzed by flow cytometry. (C and D) Splenocytes (C) and purified B cells (D) were co-incubated with DRibbles or lysate respectively for 7 days. IgM in the supernatants was analyzed by ELISA. (E) Purified B cells were co-incubated with DRibbles, tumor cell lysate or LPS for 3 days. Cytokines including IL-6, IL-10, TNF-αα in the supernatants was analyzed by ELISA. (CM indicated complete medium). Results represent three independent experiments.</p

Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy

A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al₂O₃ nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al₂O₃–Vx3) via a chemical linker. The functionalized α-Al₂O₃–Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al₂O₃–Vx3-UPs than the physical mixture of α-Al₂O₃ and Vx3-UPs (α-Al₂O₃/Vx3-UPs). Laser confocal microscopy images revealed that α-Al₂O₃–Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al₂O₃–Vx3-UPs to CD8⁺ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al₂O₃–Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al₂O₃/cell lysate or α-Al₂O₃/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al₂O₃ as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines

Additional file 2: Figure S1. of A pilot study of an autologous tumor-derived autophagosome vaccine with docetaxel in patients with stage IV non-small cell lung cancer

CONSORT diagram. (DOCX 26 kb