Lipid nanoparticles as a tool to dissect dendritic cell maturation pathways

Abstract

Depending on how antigens are being decoded by dendritic cells (DCs), their acquisition will induce a homeostatic or immunogenic maturation program. This determines how antigens are being presented and whether DCs instruct T cells to induce tolerance or immunity. So far, the field lacks proper tools to distinguish the two maturation states. By using a lipid nanoparticle (LNP)-based approach and cellular indexing of transcriptomes and epitopes sequencing analysis, we designed a flow cytometry panel and transcriptional profiling tools to reliably annotate the two DC maturation states. The data corroborate that uptake of empty (or peptide-containing) LNPs induces homeostatic maturation in DCs, while uptake of Toll-like receptor ligand-adjuvanted (or mRNA-containing) LNPs induces immunogenic maturation, yielding distinct T cell outputs. This reveals that LNPs are not decoded as "dangerous" by DCs, and that the cargo is essential to provide adjuvant activity, which is highly relevant for the targeted design of LNP-based therapies.

Keywords: CITE-sequencing; CP: Immunology; Toll-like receptor; adjuvanticity; dendritic cells; homeostatic maturation; immunity; immunogenic maturation; lipid nanoparticle; maturation; tolerance.

Graphical abstract

Figure 1

Uptake of non-cargo-loaded LNPs drives activation of a homeostatic cDC1 maturation program (A) Experimental set-up. For details, see materials and methods. (B) Non-integrated UMAP plot featuring the combined treatment and time point annotation of the nine conditions together. Subsequently, the UMAP is split per condition. (C) Non-integrated UMAP plot featuring the SS condition with cDC1 maturation stage annotation. (D) Harmony UMAP plot featuring the cDC1 maturation stage annotation of the nine conditions integrated together. We refer to the material and methods section for more information on the “other cDC1” clusters. (E) MDS plot from the muscat pseudobulk DS analysis showing the level of similarity between pseudobulk cluster-sample instances of the CITE-seq. Only the EM cluster was included for the 2-h samples and the LM cluster for the 8-h samples. For the SS sample, three clusters were included: immature, EM, and LM. n = 4 for all conditions, except CpG-LNP at 8 h (n = 3). EM: early mature; LM: late mature.

Figure 2

Common and distinct transcriptomic profiles mark homeostatic and immunogenic mature cDC1s (A and B) Triwise plots featuring the relative expression of genes in three separate conditions. (A) Comparison between immature cDC1s in SS and LM cDC1s 8 h after injection with eLNP or pIC-LNP. (B) Comparison between LM cDC1s 8 h after injection with eLNP injection, pIC-LNP, or CpG-LNP. For all details, see materials and methods. (C) Plot showing the average mRNA expression (Seurat module score) for a selection of ISGs. (D) Scatterplot indicating the type of DC maturation of various gene lists available in the literature. The maturation type, plotted on the x axis, is a score calculated as the difference in overlap between each literature gene list and the top 200 uniquely upregulated immunogenic and homeostatic cDC1 maturation genes, according to adjusted p value, respectively, in our CITE-seq data (for details on the calculation of the score, see Figure S2C). The log10 transformation of the literature gene list length determines the position on the y axis, indicative of the number of genes represented in the gene list. Literature gene lists with a score < −5 are colored in red (immunogenic), between −5 and 5 are colored in gray (undefined), and > 5 are colored in green (homeostatic). The gene lists include the names used in the original papers and are labeled with basic information about the type of DC maturation, the manuscript, the sequencing technology, and the tissue. BMDC: bone marrow-derived dendritic cell; EM: early mature; LM: late mature, SI PP: small intestinal Peyer’s patches.

Figure 3

Different transcriptional programs drive the homeostatic and immunogenic maturation programs (A–D) Heatmap of TF activity during cDC1 maturation analyzed using DoRothEA. The color scale of the heatmap represents the scaled TF activity (inferred from mRNA expression of TF targets). The columns represent the clusters from the conditions as indicated. For all details, see materials and methods. (E) Percentage of myeloid cells and lymphoid cells in the spleen of IKK2^fl/fl (n = 4) and IKK2 ^fl/flXcr1-Cre (IKK2ΔcDC1 mice) (n = 5) mice. (F) Percentage of CCR7⁺ cDC1s and cDC2s in the spleen of IKK2^fl/fl and IKK2ΔcDC1 mice (left). Percentage of CD63⁻CCR7⁺ (EM) and CD63⁺CCR7⁺ (LM) cDC1s in the spleen of IKK2^fl/fl and IKK2ΔcDC1 mice (right). Two-way ANOVA corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. (G) Cell number of CCR7⁺ cDC1s and cDC2s in the spleen of IKK2^fl/fl and IKK2ΔcDC1 mice (left). Cell number of CD63⁻CCR7⁺ (EM) and CD63⁺CCR7⁺ (LM) cDC1s in the spleen of IKK2^fl/fl and IKK2ΔcDC1 mice (right). Two-way ANOVA corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. (H) Relative expression of Ikk2, Ccr7, Fscn1, Ccl22, Cd40, Cd80, and Cd86 in sorted CCR7⁻ and CCR7⁺ cDC1s from the spleen of IKK2^fl/fl and IKK2ΔcDC1 mice (n = 5), measured by RT-qPCR. The expression was normalized to housekeeping genes Sdha and Ywhaz. Two-way ANOVA corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. The mean ± SEM is shown in (E–H). Representative of two experiments for (F) and (G). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗and p < 0.0001; not significant is not shown. EM: early mature; LM: late mature.

Figure 4

Different cell surface markers can be used to distinguish the homeostatic from the immunogenic maturation state in cDCs (A) Triwise plot of the CITE-seq data featuring the relative expression of surface proteins (ADT) in LM cDC1s 8 h after injection with eLNP pIC-LNP and CpG-LNP. For details, see materials and methods. (B) Mean fluorescence intensity (MFI) of a selection of markers in the CCR7⁻ and CCR7⁺ populations of splenic cDC1s in SS (n = 3), cDC1s 12 h after eLNP injection (n = 4), and cDC1s 12 h after pIC-LNP injection (n = 4). The statistics are shown only for the comparison between eLNP and pIC-LNP groups. Two-way ANOVA corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. (C) Representative contour plots showing the expression of MHC class II versus CCR7 before the dashed line (left) and the expression of selected immunogenic markers on the y axis versus selected homeostatic markers on the x axis. (D) Experimental set-up of the T. gondii experiment showing that 500 T. gondii tachyzoites were injected intraperitoneally 6 days before analysis of the spleen. (E) Percentage of CCR7⁺ cDC1s in control mice (n = 5) or mice infected with T. gondii (n = 5). (F) MFI of a selection of markers showing the CCR7⁻ and CCR7⁺ populations of cDC1s of control mice (n = 5) and mice infected with T. gondii (n = 5). Two-way ANOVA corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. (G) Experimental set-up of the MC38 colon carcinoma model showing that MC38 cells were subcutaneously injected 12 days before intratumoral treatment with either eLNPs, pIC-LNPs, or control (not injected). After 8 h, the tumor and tdLN were analyzed. (H) MFI of CD80 and CD63 of CCR7⁻ and CCR7⁺ cDC1s in the tumor and tdLN. Two-way ANOVA corrected for multiple testing by Tukey’s multiple comparisons test with a single pooled variance. The mean ± SEM is shown in (B), (E), (F), and (H). Representative of two experiments for (B), (D), (E), (F), (G), and (H). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001; not significant is not shown.

Figure 5

Homeostatic and immunogenic mature DCs provoke distinct T cell outputs (A) Histogram plots showing CXCL16, CXCL9, and CCL5 expression of splenic CCR7⁻ and/or CCR7⁺ cDC1 populations of representative samples in SS, 8 h after injections with eLNP, or pIC-LNP. MFI plots show the expression of the CCR7⁻ and/or CCR7⁺ populations of the three conditions (n = 4 for all). One-way ANOVA corrected for multiple testing by Tukey’s multiple comparisons test with a single pooled variance (for CXCL9 and CCL5). Brown-Forsythe and Welch ANOVA corrected for multiple testing by Dunnett’s T3 multiple comparisons test with individual variances (for CXCL16). (B) MFI of CD80 of CCR7⁻ and/or CCR7⁺ cDC1s in SS (n = 4) or 12 h after injection with eLNPs made with the ionizable lipid ALC-0315 (n = 5), eLNPs made with the ionizable lipid SM-102 (n = 5), LNPs containing OTI and OTII peptide (OTI/OTIIpeptide-LNPs) (n = 5), mRNA-LNPs (n = 3), or pIC-LNPs (n = 5). All LNPs were made with the ionizable lipid ALC-0315 except otherwise indicated. Kruskal-Wallis test corrected for multiple testing with Dunn’s multiple comparison test. (C) Experimental set-up for data shown in (D), (E), and (F): CD45.2 acceptor mice were injected with 0.5 million CD45.1.2 OTII cells. Two days later, the mice were injected with different LNPs, and at two time points, the OTII cells were checked in the blood by flow cytometry. (D) Percentage of OTII cells of CD4 T cells in the blood at days 7 and 12 of control mice (n = 5) or after injection of eLNPs (n = 5), OTIIpeptide-LNPs (n = 5), and OTIIpeptide-pIC-LNPs (n = 4). Two-way ANOVA, Tukey’s multiple comparisons test with single pooled variance. (E) Percentage of Foxp3+ OTII cells in the blood 7 and 12 days after injection of OTII peptide-LNPs and OTII-peptide-pIC-LNPs. (F) Percentage of CD44⁺CD62L⁺ and CD44⁺CD62L⁻ OTII cells in the blood at day 7 (n = 5 for OTII peptide-LNPs and n = 4 for OTII peptide-pIC-LNPs) and day 12 (n = 4 for OTII peptide-LNPs and n = 3 for OTII peptide-pIC-LNPs) after LNP injections. Mixed-effects model corrected for multiple testing by Sídák’s multiple comparisons test with a single pooled variance. Representative contour plots (right) showing the expression of CD44 versus CD62L, gated on OTII cells. For (E and F), two datapoints were omitted on day 12 due to low counts of OTII cells. (G) Experimental set-up for data shown in (H), (I), and (J), indicating that CD45.2 acceptor mice were injected with 0.3 million CD45.1.2 OTI cells. One day later, the mice were injected with different LNPs. Three days later, the OTI cells were checked in the blood by flow cytometry. At day 9, the mice were injected with SIINFEKL-lentivirus, and at day 13, the blood was checked again, and an ex vivo cytotoxicity assay was performed. (H) Percentage of OTI cells of CD8 T cells in the blood at days 3 and 13 (left) of control mice (n = 4) or after injection of eLNPs (n = 5), OTI peptide-LNPs (n = 6) and OTI peptide-pIC-LNPs (n = 6). Two-way ANOVA, corrected for multiple testing by Tukey’s multiple comparisons test with a single pooled variance. Fold-change ratio (day13/day 3) of OTI cells in the blood (right). One-way ANOVA, corrected for multiple testing by Tukey’s multiple comparisons test with a single pooled variance. (I) Percentage of CD44⁺CD62L⁺, CD44⁺CD62L⁻, and CD44⁻CD62L⁻ OTI cells in the blood at day 13 (n = 6 for both OTII peptide-LNPs and OTII peptide-pIC-LNPs). Two-way ANOVA with Geisser-Greenhouse correction, corrected for multiple testing by Sídák’s multiple comparisons test with individual variances. (J) OTI cytotoxicity was measured as the ratio of live CTV-labeled (WT thymocytes) and CTR-labeled (OVA thymocytes) after co-culture with splenocytes. Kruskal-Wallis test, corrected for multiple testing by Dunn’s multiple comparisons test. The mean ± SEM is shown in (A), (B), (D), (E), (F), (H), (I), and (J). Representative of two experiments for (A), (B), (C), (D), (E), (F), (G), (H), and (I). For (J), n = 1. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001; not significant is not shown.