Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response

Abstract

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Copyright © 2019 by The American Association of Immunologists, Inc.

Figures

FIGURE 1.

R-DOTAP formulations containing multiple tumor-associated Ags induce quantitatively and qualitatively superior CD8 T cell responses. Groups of AAD mice (n = 6) were s.c. injected with a human MUC1 multipeptide formulation containing the indicated MUC1 CD8 T cell epitopes formulated with R-DOTAP or other adjuvants. Mice were injected on day 0 and day 7, and Ag-specific CD8 T cell responses in spleen were assessed 7 d after the second injection. (A) ELISPOT analysis measuring the number of V1A, V2A, C1A, and C2A specific IFN-γ–producing cells in spleens from mice injected with an R-DOTAP–MUC1 formulation. (B) ELISPOT analysis measuring the number of V1A and V2A specific IFN-γ–producing cells in spleen from mice injected with R-DOTAP, IFA-Cyt, Montanide, or sucrose formulations containing MUC1 peptides. (C) Intracellular cytokine staining of VIA- or PMA/ionomycin–stimulated spleen cells following immunization with the indicated formulations. Graph represents the percentage of IFN-γ–producing cells among total CD8 T cells. Pie charts represent the percentage of cells producing additional cytokines IL-2 and TNF-± among total IFN-γ–producing cells. Data represent the mean ± SEM of n = 5–6 mice in each group. Experiments were repeated at least three times with similar results. **p < 0.05.

FIGURE 2.

R-DOTAP efficiently alters effector to suppressor T cell ratio, promoting tumor regression. (A) Groups of C57BL/6J mice (n = 5) were s.c. injected with Ag KF18 plus R-DOTAP (A+R), KF18 plus R-DOTAP plus GM-CSF (A+R+G), KF18 plus GM-CSF (A+G), or KF18 plus sucrose (A+S) and compared with naive (N) mice. Mice were boosted on day 7 with the same formulations and analyzed by IFN-γ ELISPOT on day 14 using RF9 as a stimulatory peptide. (B) Mice (n = 10) were implanted s.c. with 1 × 105 TC-1 tumor cells and, when the tumors reached an average diameter of 4–5 mm (day 11), were administered a single dose of formulation containing R-DOTAP mixed with HPV mix (R-DOTAP.HPV), R-DOTAP plus HPV mix plus GM-CSF (R-DOTAP.HPV.GM-CSF), HPV mix plus GM-CSF (HPV.GM-CSF), or HPV mix plus sucrose (HPV.suc), and tumor growth was monitored. (C-G) Parallel groups of tumor-bearing mice (n = 5), treated as in (B), were euthanized 20 d after tumor implant when some groups showed initial signs of regression, and tumors were processed to assess tumor-infiltrating lymphocytes (TILs). Live CD45+CD3+ cells were gated, and the percentages of CD8+ (C), CD4+ (D), and RF9-Db dextramer+ cells among CD8+ TILs (E) were determined. (F) The CD8/CD4 ratio and (G) the T regulatory cell (Treg) (CD3+CD4+Foxp3+CD25+) to RF9-Db dextramer binding (Treg/RF9 ratio) were calculated. Data represent the mean ± SEM from each group (n = 5), and experiments were repeated at least three times with similar results. **p < 0.05.

FIGURE 3.

A single peptide and R-DOTAP immunotherapy can regress large TC-1 tumors and induce durable immunity. Mice implanted s.c. with 1 × 105 TC-1 tumor cells were injected with one dose of KF18 and R-DOTAP when the tumors reached the average diameters shown, ranging from 4 to 9 mm (A-C). (D) Mice were injected with R-DOTAP without peptide. (E) Naive mice received no treatment. (F) Mice bearing primary tumors (4–5 mm) were injected with one dose of an R-DOTAP formulation containing KF18 peptide on day 7 (open black circles), and after complete tumor regression, the mice were rechallenged with a second dose of 1 × 105 TC-1 tumor cells. Naive mice receiving a tumor implant at the time of primary injection are shown as gray diamonds, and naive mice receiving a tumor implant at the time of rechallenge are shown as gray triangles. Data represent tumor measurements of each mouse (n = 8–12 mice) over a period of 85 d.

FIGURE 4.

R-DOTAP synergizes with anti-mouse PD1 treatment to significantly alter B16 melanoma tumor growth in vivo. (A) Groups of C57BL/6J mice (n = 5) were s.c. injected with Trp2-encapsulated R-DOTAP (Trp2+R-DOTAP) nanoparticles or Trp2 mixed in sucrose buffer (Trp2+Sucrose) on day 0 and boosted on day 7. Trp2-specific CD8 T cell responses in spleen were assessed 7 d after the second injection by ELISPOT assay. (B-D) Mice were implanted s.c. with 1 × 105 B16F10 melanoma cells and received two doses (days 5 and 12 after tumor implant) of Trp2 plus R-DOTAP, Trp2 plus R-DOTAP plus anti-PD1, or anti-PD1 alone. For anti-PD1 treatment, each mouse received five doses of 200 μg of anti-mouse PD1 Ab delivered i.p. at 3-d intervals starting on day 5 after tumor implant. (B) Mean tumor volume ± SEM (n = 5) in treated or naive mice. (C) Survival over the course of the study. (D) Tumor growth kinetics in each individual mouse. Experiments were repeated three times with similar results. **p < 0.05.

FIGURE 5.

R-DOTAP enhances peptide cross-presentation in vitro and in vivo. (A) BMDCs were incubated with Alexa 647–conjugated OVA admixed with sucrose or R-DOTAP nanoparticles for the indicated times, and the association of OVA with BMDCs was determined by flow cytometry and represented as the geometric mean fluorescence intensity. (B) BMDCs were incubated with DQ-OVA, DQ-OVA plus R-DOTAP (OVA+RDOTAP), or DQ-OVA plus LPS (OVA+LPS) for 60 min, washed, and analyzed by flow cytometry. DQ-OVA processing was measured by assessing the fluorescence in the FITC channel (FL1H) and the fluorescence in the PE-channel (FL2H) representing OVA processing. (C) BMDCs were pulsed for 10 min with the indicated concentrations of OVA241–270 peptide admixed with sucrose (green circles) or R-DOTAP (red squares) and cocultured with B3Z cells overnight. Production of lacZ by OVA peptide-stimulated B3Z was measured using a lacZ colorimetric assay and displayed as relative absorbance compared with untreated B3Z cells. (D-F) C57BL/6 or BALB/c mice were adoptively transferred with CFSE-labeled OT1 or DO11.10 spleen cells and, after 24 h, injected s.c. with 1 μg OVA admixed with 4 mm R-DOTAP (OVA+R-DOTAP) or sucrose buffer (OVA). (D) The total number of cells in the draining popliteal LNs in each treated mouse were enumerated. (E) Total CFSE-labeled cells per LN. (F) Total Ag-specific CD8 T cell expansion was measured by CFSE dilution assay and quantitating cells that have undergone at least one division (M2) or no divisions (M0) using a flow cytometer. n = 4 mice per group. Results represent the mean ± SD. All experiments were repeated at least three times with similar results. *p < 0.05, **p < 0.05.

FIGURE 6.

R-DOTAP administration induces production of type I IFN in the DLN in vivo. (A) Groups of four C57BL/6 mice were injected with R-DOTAP, PBS, or LPS at the nape of the neck, and draining axillary and brachial LNs were harvested from each mouse after 4 or 24 h. CD11c+ cells from pooled LNs of each individual mouse were sort-purified, and relative gene expression was analyzed using Nanostring technology. Shown are mean RNA expression levels for genes from the R-DOTAP or LPS groups that showed >0.5-fold difference relative to PBS treatment. (B) BMDCs from IFNAR−/− mice were stimulated with the indicated concentrations of R-DOTAP nanoparticles or LPS for 24 h, and type I IFN production was measured using an IFN-α/β reporter assay. (C) Mice were injected with R-DOTAP or sucrose in the footpad, DLN were harvested at the indicated times and enzymatically digested, and total cell number was enumerated. (D) Wild-type (WT) or IFNAR−/− mice were administered R-DOTAP or sucrose (Suc.) in the footpad, and total cells per DLN were quantitated after 24 h. (E) DLN from mice described in (D) were analyzed for the percentage of CD69+ NKP36+ NK cells or (F) the percent of CD69+ CD3+ T cells. Experiments were repeated at least three times with similar results.

FIGURE 7.

R-DOTAP mediates its CTL-inducing effects by activating type I IFN in a Myd88-dependent manner. (A) Wild-type (WT) or IFNAR−/− mice were injected on days 0 and 7 with the OVA SL9 peptide admixed with R-DOTAP (SL9+R-DOTAP), SL9 peptide emulsified with CFA (SL9+CFA), or SL9 in sucrose (SL9+Suc), and ELISPOTs were performed against the SL9 peptide on day 14. (B) WT, TRIF−/−, or MYD88−/− mice were injected on days 0 and 7 with the RF9 peptide encapsulated in R-DOTAP nanoparticles (RF9.R-DOTAP), and ELISPOTs were performed against the RF9 peptide on day 14. (C) WT, STING−/−, IFNAR−/−, or Myd88−/− mice were injected on days 0 and 7 with RF9 peptide encapsulated in R-DOTAP nanoparticles (RF9.R-DOTAP), and ELISPOTs were performed against the RF9 peptide on day 14. Data represent the mean ± SD of peptide-specific IFN-γ–producing cells in spleens from treated mice (n = 5). (D) HEK-Blue null, HEK-Blue human TLR9, and (E) HEK-Blue human TLR7 reporter cells were stimulated for 24 h with the indicated concentration of R-DOTAP nanoparticles, ODN2006 or ODN2395 (D), R848 (E), or PMA; the cell supernatants were assayed for SEAP activity using QUANTI-Blue reagent; and the background subtracted relative absorbance in triplicate wells were plotted. Data represent mean absorbance ± SEM of triplicate cultures, and all experiments were repeated at least two times with similar results. **p < 0.05.