Lipid nanoparticles (LNP) induce activation and maturation of antigen presenting cells in young and aged individuals

Abstract

Herein, we studied the impact of empty LNP (eLNP), component of mRNA-based vaccine, on anti-viral pathways and immune function of cells from young and aged individuals. eLNP induced maturation of monocyte derived dendritic cells (MDDCs). We further show that eLNP upregulated CD40 and induced cytokine production in multiple DC subsets and monocytes. This coincided with phosphorylation of TANK binding kinase 1 (pTBK1) and interferon response factor 7 (pIRF7). In response to eLNP, healthy older adults (>65 yrs) have decreased CD40 expression, and IFN-γ output compared to young adults (<65 yrs). Additionally, cells from older adults have a dysregulated anti-viral signaling response to eLNP stimulation, measured by the defect in type I IFN production, and phagocytosis. Overall, our data show function of eLNP in eliciting DC maturation and innate immune signaling pathways that is impaired in older adults resulting in lower immune responses to SARS-CoV-2 mRNA-based vaccines.

Fig. 1. eLNP treatment promotes robust innate immune response and maturation.

a Activation status of monocyte-derived DCs as measured by CD40 expression after 24 h of 15 μg/mL (total lipids, or~7.5 μg/mL ionizable lipid) empty LNP (eLNP) stimulation (yellow). n = 18. b Innate cell cytokine expression after 24 h eLNP stimulation (yellow) compared to that in unstimulated (media) cells (grey) of MDDCs. n = 18 for each group as measured by Luminex. c Activation status of DC and monocyte subsets from PBMCS as measured by CD40 expression after 24 h of eLNP stimulation (yellow). n = 18. d Dynamics of cytokines from PBMCS at 0, 6, and 24 h after eLNP stimulation. n = 18 as measured by Luminex. e Dynamics of cytokines from monocytes at 0, 6, and 24 h after eLNP stimulation. n = 18 as measured by Luminex. Each individual circle represents one individual subject. Data was analyzed either using non-parametric Mann Whitney T-test or by one-way ANOVA followed by Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. IRF7/TBK-1 axis is important for eLNP-induced innate response.

Human PBMCs from healthy donors were either stimulated with 15 μg/mL (total lipids, or ~7.5 μg/mL ionizable lipid) eLNP for 15 min, 45 min, 6, or 24 h. For unstimulated and control conditions refer to Fig. 1. Cells were permeabilized, fixed, and stained for (a) phosphorylated interferon response factor 7 (IRF7) transcription factor or (b) phosphorylated TBK-1. After gating on monocyte and DC subsets, the geometric mean intensity (MFI) was measured using phosflow cytometry. Each individual circle represents one individual subject. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA.

Fig. 3. Phagocytosis is induced in cDC2, cDC1, CD14^dim CD16⁺ monocytes in response to eLNP stimulation.

Phagocytosis measured using fluorescent beads and multiparametric flow cytometry. PBMCs were incubated overnight with stimulus (15 μg/mL (total lipids, or ~7.5 μg/mL ionizable lipid) eLNP or plain medium) and with beads for a further 3 h. a FACS plot examples of phagocytosis assay as geometric mean fluorescent intensity (MFI) by flow cytometry. b Phagocytosis after 24 h eLNP stimulation (yellow) compared to that in unstimulated cells (grey) of PBMCS. n = 18 for each group. Each individual circle represents one individual subject, n = 18. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by or non-parametric Mann Whitney T-test.

Fig. 4. eLNP treatment promotes TGF-B secretion.

a Dynamics of TGF-β secretion from PBMCS at 0, 6, and 24 h after eLNP stimulation. n = 18. Data were combined from at least two independent experiments. Each individual circle represents one individual subject. Data are from one independent experiment (a) One-way ANOVA followed by Tukey’s test was applied in a. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5. eLNP treatment promotes monocyte-derived DC maturation and the secretion of pro-TFH cytokines that are also diminished between young and older participants.

a–d Monocyte-derived dendritic cells (MDDCs) differentiated in vitro from monocytes of healthy human PBMCs from older and younger donors, were treated with 15 μg/mL (total lipids, or ~7.5 μg/mL ionizable lipid) eLNP for 24 h and compared to unstimulated cells (just media) as controls. MDDCs were harvested and stained for surface HLA and costimulatory molecule expression and analyzed by flow cytometry. Values are shown as the frequencies of (a) CD83, (b) CD40, (c) CD86 (d) HLA-DR in the live, CD3- CD19- CD11c + MDDC population. Supernatants were also harvested, and selected cytokines were measured with Luminex after 24 h of stimulation (a–d). Nine samples per group were measured, except 24 h LPS where only three samples per group were analyzed. Results are expressed as mean ± SEM of 2 independent experiments (n = 9). Each individual circle represents one individual subject. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA or non-parametric Mann Whitney T-test.

Fig. 6. Phagocytosis is induced in response to eLNP stimulation.

a, b Phagocytosis is induced in cDC2, cDC1, CD14^dim CD16⁺ monocytes. Phagocytosis measured using fluorescent beads and multiparametric flow cytometry. PBMCs were incubated overnight with stimulus and with beads for a further 3 h. Dotted grey line represents an example of a young donor unstimulated. Solid grey represents an example of a young donor stimulated with eLNP. Dotted blue line represents an example of an older donor unstimulated. Solid blue represents an example of an older donor stimulated with eLNP. Each individual circle represents one individual subject. n = 9, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by non-parametric Mann Whitney T-test.

Fig. 7. eLNP treatment results in differential TGF-β secretion.

a TGFB secretion after 24 h eLNP stimulation compared to that in unstimulated cells of MDDCs. n = 9. Data were combined from at least two independent experiments. b Dynamics of TGF-B secretion from PBMCS at 0, 6, and 24 h after eLNP stimulation. n = 9 for each group. Data are from one independent experiment (a,b) Each individual circle represents one individual subject. One-way ANOVA followed by Tukey’s test was applied in a. Non-parametric Mann Whitney T-test was applied in b. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8. Lipid nanoparticles (LNP) can activate the innate immune response sans mRNA (eLNP).

eLNP will upregulate co-stimulatory makers on the surface of monocytes, DCs, and PBMCs that are necessary for T cell activation and antigen-processing and presentation. eLNP will then induce a productive humoral and cellular response. In older adults, however, eLNP upregulates the same co-stimulatory but to a much lesser degree while also upregulating PD-L1, an inhibitory marker that was all but absent in younger adults. Addition of eLNP also induced TGF-B production in older adults. Taken together, our study shows a mechanism that accounts for the decreased protection due to vaccination in older adults.