Research ArticleVACCINES

Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation

See allHide authors and affiliations

Science Immunology  21 Jul 2017:
Vol. 2, Issue 13, eaal1329
DOI: 10.1126/sciimmunol.aal1329
  • Fig. 1 LV pseudotransduction delivers proteins and activates DCs.

    (A) Representative fluorescence-activated cell sorting (FACS) plots of mouse BMDCs that were treated with LV-GFP(V), LV-GFP(S), LPS, or no vector (NV) and analyzed for expression of GFP, CD86, and I-Ab. GFP geometric MFI was measured immediately after LV spin inoculation, and CD86 and I-Ab expression was measured 24 hours after LV treatment. (B) GFP, CD86, and I-Ab expression of LV-treated BMDCs was measured over 48 hours. (C) Representative FACS plots of mouse BMDCs, human moDCs, and 293T cells that were incubated with tenofovir (TFV; 40 μM), efavirenz (EFV; 80 μM), or no drug (ND) 6 hours before treatment with LV-GFP(V) and then analyzed 24 hours later. (D) Graph depicts the GFP MFI of BMDCs, moDCs, and 293T cells from (C). (E) Mouse BMDCs were incubated with or without cycloheximide (CHX; 50 μg ml−1) 1 hour before treatment with LV-GFP(V), and then, GFP MFI was presented relative to those BMDCs receiving no LV with or without cycloheximide. (F) Western blot analysis of GFP of lysates from LV-GFP(V) and LV expressing OVA pseudotyped with VSV-G [LV-OVA(V)] and purified GFP protein (40 ng). (G) Mouse BMDCs were treated as in (A) and analyzed for the amount of IL-6 and IL-12/23 in the supernatant by ELISA 24 hours after LV treatment. (H and I) Mouse BMDCs (H) and human moDCs (I) were treated as in (C) and analyzed for expression of CD86, I-Ab, or amount of IL-12/23 and/or IL-6 in the supernatant 24 hours after LV treatment. Data are representative of two (A to D and G to I) or three (E and F) independent experiments. Results are shown as mean ± SEM (B, D, E, G, and H). n.s., not significant. P > 0.05; ***P < 0.001 [one-way ANOVA (D and H) and unpaired Student’s t test (E and G)].

  • Fig. 2 LV envelope is responsible for DC activation.

    (A) Schematic of components of LV and VLPs. (B) Western blot analysis for GFP, VSV-G, and p24 on LV and VLP lysates. (C) Western blot analysis detecting OVA in the lysate of the following SVGmu-pseudotyped vectors: LV carrying OVA (LV-OVA), VLP carrying OVA (VLP-OVA), and VLP-carrying OVA deficient of gag (VLP-OVAΔgag). Vectors were treated or not treated with proteinase K, which was inactivated with PMSF before vector lysis. To verify whether proteinase K degradation was effective, we used soluble OVA as a control. (D and E) Mouse BMDCs were treated with VSV-G or SVGmu-pseudotyped LVs and VLPs and then analyzed at 24 hours for GFP, CD86, and I-Ab expression by flow cytometry (D) and for the amount of IL-6 and IL-12/23 in the cell supernatant by ELISA (E). (F) Human moDCs were treated with LV-GFP(V) or VLP carrying GFP deficient of gag [VLP-GFPΔgag(V)] and analyzed at 24 hours for GFP, CD86, and human MHC II molecule human lymphocyte antigen-D–related (HLA-DR) expression by flow cytometry. Data are representative of three (B and C) or two (D to F) independent experiments. Results are shown as mean ± SEM. P > 0.05; **P < 0.005; ***P < 0.001 (unpaired Student’s t test).

  • Fig. 3 LV DNA, genome, and capsid are not required for DC activation and CD8+ T cell priming in vivo.

    (A) Wild-type mice received homologous prime-boost vaccination of SVGmu-pseudotyped LV-OVA with or without RTI or VLP-OVA (n = 8 mice per group). Representative FACS plots show OVA-tetramer+ cells gated on CD8+ T cells from the blood at 7 and 10 days after primary immunization (left). Graph depicts percentages of OVA-tetramer+ CD8+ T cells from the blood of immunized and unimmunized mice over time (black arrow, boost) (right). (B) Representative FACS plots show expression of CD62L and CD44 on OVA-tetramer+ CD8+ T cells from immunized mice compared with naïve CD8+ T cells from unimmunized mice at 7 days after boost (left). Graphs depict percentages of CD62Llo and CD44hi OVA-tetramer+ cells, with each symbol representing an individual mouse and horizontal bar indicating the mean (right). (C) Seven weeks after boost, mice were injected with 5 × 106 OVA-expressing E.G7 thymoma tumor cells and 5 × 106 EL4 (control) non–OVA-expressing EL4 thymoma tumor cells on opposing legs, and tumor sizes were measured. (D to F) Wild-type mice were homologously prime-boosted with LV-OVA, VLP-OVA, or capsid-less VLP-OVAΔgag. OVA-tetramer+ cells from the blood were analyzed as in (A) (n = 8 mice per group) at 7 days after boost (left) and over time (right) (D). CD62L and CD44 expression of OVA-tetramer+ CD8+ T cells from immunized mice compared with naïve CD8+ T cells from unimmunized mice at 7 days after boost were measured as in (B) (E). Mice were injected with tumor cells, as in (C), and tumor sizes were measured (F). (G) Wild-type mice were immunized with LV-OVA, LV encoding OVA carrying GFP (LV-GFPgene-OVAprotein), or VLP-OVA (n = 8 mice per group), and OVA-tetramer+ CD8+ T cells from the blood were measured over time. Statistical comparisons were made between the LV-GFPgene-OVAprotein– and VLP-OVA– or LV-OVA–immunized mice. Data are representative of two independent experiments (A to G). Results are shown as mean ± SEM (A, C, D, F, and G). P > 0.05; **P < 0.005; ***P < 0.001 (unpaired Student’s t test).

  • Fig. 4 LV activation of DCs and subsequent CD8+ T cell priming are dependent on STING and cGAS but not on MyD88, TRIF, or MAVS.

    (A and B) BMDCs from mice singly or doubly deficient in MyD88, TRIF, and MAVS were treated with LV-GFP(V) or LV-GFP(S) and analyzed at 24 hours for expression of CD86 and I-Ab by flow cytometry. (C to F) Mice deficient in MyD88, TRIF, MAVS, STING, or cGAS were immunized with LV-OVA. Unimmunized wild-type (WT) mice were injected with PBS. OVA-tetramer+ cells gated on CD8+ T cells from the blood were demonstrated on representative FACS plot at 10 days after primary immunization (C) or measured over time (D to F) (left). Statistical comparisons were made between the OVA-tetramer+ CD8+ T cell response of the LV-immunized wild-type mice and that of the LV-immunized mutant mice. CD62Llo and CD44hi OVA-tetramer+ CD8+ T cells from LV-immunized mutant and wild-type mice were compared with naïve CD8+ T cells from unimmunized mice at 10 days on representative FACS plots (D to F) (middle) or by group with each symbol representing an individual mouse and horizontal bar indicating the mean (D to F) (right). n = 6 mutant immunized mice per group; n = 4 wild-type immunized and unimmunized mice per group (C and D). n = 6 mice in Tmem173−/− immunized and unimmunized wild-type groups; n = 10 mice in wild-type immunized group (E). n = 8 per group (F). Data are representative of three (A and B) or two (C to E) independent experiments or pooled from two independent experiments (F). Results are shown as mean ± SEM (A, B, and D to F). P > 0.05; *P < 0.05; **P < 0.005 [one way-ANOVA (A, B, and D) and unpaired Student’s t test (E and F)].

  • Fig. 5 VLPs activate DCs and antigen-specific CD8+ T cells via the STING and cGAS pathway.

    (A and B) BMDCs from Tmem173−/− and cGAS−/− mice were treated with VLP-GFP(V) or VLP-GFP(S) and analyzed at 24 hours for CD86 and I-Ab expression by flow cytometry. (C and D) Tmem173−/− and cGAS−/− BMDCs were treated with VLP-OVA pseudotyped with VSV-G [VLP-OVA(V)] or SVGmu [VLP-OVA(S)] and then cocultured with OT-1 CD8+ T cells for 24 hours. Representative FACS plots show expression of the T cell activation marker CD69 among the OT-1 CD8+ T cells (C and D) (left). Graph depicts CD69 MFI of the OT-1 CD8+ T cells (C and D) (right). (E to H) Tmem173−/−, cGAS−/−, and wild-type mice were homologously prime-boosted with SVGmu-pseudotyped VLP-OVA (n = 8 per group). OVA-tetramer+ cells gated on CD8+ T cells from the blood were demonstrated on representative FACS plot at 10 days after primary immunization (E and G) (left) or measured over time (E and G) (right) (black arrow, boost). Statistical comparisons were made between the OVA-tetramer+ CD8+ T cell response of the VLP-immunized wild-type and VLP-immunized mutant mice. CD62Llo and CD44hi OVA-tetramer+ CD8+ T cells from LV-immunized Tmem173−/−, cGAS−/−, and wild-type mice were compared with naïve CD8+ T cells from unimmunized wild-type mice at 10 days on representative FACS plots (F and H) (left) or by group with each symbol representing an individual mouse and horizontal bar indicating the mean (F and H) (right). Data are representative of two independent experiments (A to H). Results are shown as mean ± SEM (A to E and G). P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.001 (unpaired Student’s t test).

  • Fig. 6 Viral fusion is required for DC activation.

    (A) BMDCs from wild-type mice were incubated with chloroquine (CQ) at 25, 75, 100 μM (wedges) or with no drug 1 hour before treatment with LV or VLPs and analyzed at 24 hours for CD86 and I-Ab expression by flow cytometry. (B) Fluorescence microscopy was used to analyze GFP expression in wild-type mouse BMDCs treated with fusion-competent VLP-GFP(V) with or without chloroquine (100 μM) or the fusion-defective VLP, VLP-GFP(V.FD). Magnification, ×400. Scale bars, 10 μm. (C) Wild-type mouse BMDCs were treated with fusion-competent or fusion-defective LVs or VLPs carrying GFP and analyzed for CD86 and I-Ab expression by flow cytometry. (D and E) BMDCs from wild-type and Tmem173−/− mice were treated with naked (Lipo) or VSV-G–enveloped multilamellar liposomes [Lipo(V)] carrying GFP and analyzed at 24 hours for CD86 and I-Ab expression by flow cytometry. (F) Wild-type mouse BMDCs were treated with VSV-G– or SVGmu-pseudotyped VLPs with or without LY292004 (50 μM) and analyzed for CD86 expression by flow cytometry and for the amount of IL-12/23 and IL-6 in the supernatant by ELISA. (G) BMDCs from Tmem173−/−, cGAS−/−, and wild-type mice were treated with VSV-G–pseudotyped VLP or LPS (100 ng ml−1) with or without LY292004 (50 μM) and analyzed for CD86 expression by flow cytometry. Data are representative of three independent experiments (A to D) or two independent experiments (E to G). Results are shown as mean ± SEM (A and C to G). P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.001 [one way-ANOVA (C and G) and unpaired Student’s t test (D to F)].

  • Fig. 7 LV particles and VLPs contain human genomic DNA recognized by the host STING pathway.

    (A) PCR analysis of amplicons of human β-actin (ACTB) and the ampicillin resistance gene (amp) detected in the LV and VLP preparations by PCR. The cell-free supernatant collected from 293T cells transiently transfected with mock plasmid puc19 was used as a negative control. bp, base pairs. (B and C) Amplicons of human Alu element (Alu), ACTB, and VSV-G (VSIVgp4) (lane 1) were analyzed from VLP preparations (B) or genomic DNA (gDNA) (C). VLP preparations and genomic DNA were treated with lysis buffer in the presence of DNase I, leading to DNA degradation (lane 2). DNase I was inactivated with EDTA before lysis treatment (lane 3). DNase I was inactivated with EDTA, and then, VLP lysate or genomic DNA added to show DNase I was effectively inactivated (lane 4). (D) Amplicons of ACTB, Alu, and amp were analyzed on HIV-1 supernatant collected from 293T cells transiently transfected with the plasmid encoding infectious HIV-1 or 293T genomic DNA. (E) HIV-1 was passaged in primary PBMCs, and the cell-free supernatant was collected and analyzed for human ACTB by PCR. As a negative control, cell-free supernatant was collected from uninfected PBMCs. (F) Supernatant primary PBMCs passaged with HIV-1 were treated as in (B) and (C). (G and H) BMDCs from wild-type mice were treated with naked (Lipo) or VSV-G–enveloped multilamellar liposomes carrying plasmid DNA [Lipo-plasmid(V)], genomic DNA extracted from 293T cells [Lipo-gDNA(V)], or nothing [Lipo(V)] (G). BMDCs from Tmem173−/− or wild-type mice were treated with naked (Lipo) or VSV-G–enveloped multilamellar liposomes carrying plasmid DNA [Lipo-plasmid(V)], genomic DNA extracted from 293T cells [Lipo-gDNA(V)], or nothing [Lipo(V)] (H). Cells were analyzed 24 hours after treatment for CD86 and I-Ab expression by flow cytometry. (I) Wild-type mouse BMDCs were treated with LV generated from transient transfection or plasmid-free stable cell line and analyzed for CD86 expression by flow cytometry and for the amount of IL-12/23 and IL-6 in the supernatant by ELISA. Data are representative of three (A to F) or two (G to I) independent experiments. Results are shown as mean ± SEM (G to I). P > 0.05; *P < 0.05; **P < 0.005 (unpaired Student’s t test).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/13/eaal1329/DC1

    Materials and Methods

    Fig. S1. Flow cytometry gating strategies.

    Fig. S2. LV-mediated GFP expression and activation of BMDCs are dose-dependent.

    Fig. S3. Mouse bone marrow–derived CD11c+CD11b+ cells are pseudotransduced and activated.

    Fig. S4. Mouse cDCs are pseudotransduced in vivo.

    Fig. S5. LV activation of DCs is independent of TLR4 and type I IFN signaling.

    Fig. S6. Wild-type and mutant bone marrow–derived CD11c+CD11b+ cells are generated in GM-CSF culture.

    Fig. S7. VSV-G viral fusion activates PI3K.

    Fig. S8. Nonviral DNA in vector particle is primarily dsDNA, fragmented, and human genomic in origin.

    Table S1. Antibodies used in this study.

    Table S2. Primer sets used in this study.

  • Supplementary Materials

    Supplementary Material for:

    Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation

    Jocelyn T. Kim, Yarong Liu, Rajan P. Kulkarni, Kevin K. Lee, Bingbing Dai, Geoffrey Lovely, Yong Ouyang, Pin Wang, Lili Yang, David Baltimore*

    *Corresponding authors. Email: baltimo{at}caltech.edu

    Published 21 July 2017, Sci. Immunol. 2, eaal1329 (2017)
    DOI: 10.1126/sciimmunol.aal1329

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Flow cytometry gating strategies.
    • Fig. S2. LV-mediated GFP expression and activation of BMDCs are dosedependent.
    • Fig. S3. Mouse bone marrow–derived CD11c+CD11b+ cells are pseudotransduced and activated.
    • Fig. S4. Mouse cDCs are pseudotransduced in vivo.
    • Fig. S5. LV activation of DCs is independent of TLR4 and type I IFN signaling.
    • Fig. S6. Wild-type and mutant bone marrow–derived CD11c+CD11b+ cells are generated in GM-CSF culture.
    • Fig. S7. VSV-G viral fusion activates PI3K.
    • Fig. S8. Nonviral DNA in vector particle is primarily dsDNA, fragmented, and human genomic in origin.
    • Table S1. Antibodies used in this study.
    • Table S2. Primer sets used in this study.

    Download PDF

    Files in this Data Supplement:

Stay Connected to Science Immunology

Navigate This Article