Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Spatiotemporal control of mRNA delivery for enhanced APC-tropic antigen expression and immune activation (A) Female C57BL/6 mice ( n = 6) were immunized with LNP or LSE formulations containing 5 μg of OVA mRNA per mouse. At 24 h post-administration, single-cell suspensions were pooled for scRNA-seq, employing an “enrich-mix” strategy to capture immune and non-immune cell types at the injection site. T-distributed stochastic neighbor embedding (t-SNE) plots illustrated annotated cell clusters with detailed cluster annotations listed on the right. Working flow, detailed cell clusters with annotated markers are provided in . (B) OVA mRNA enrichment in each cell overlaid on the t-SNE plots. Small gray dots represent cells with no measurable OVA mRNA. (C) Antigen levels in intra- and intercellular compartments at the injection site 1 and 3 days post-immunization, determined by ELISA following separation of cellular and supernatant fractions. Female BALB/c mice ( n = 6) were intramuscularly injected with 5 μg of gE mRNA. (D) Flow cytometry analysis of immune cell recruitment at the injection site was performed in C57BL/6 mice ( n = 6) 24 h after intramuscular administration of OVA mRNA formulated with PBS, LNP, or LSE. Pie charts represent the relative proportions of recruited macrophages (Macro: CD45 + CD11b + F4/80 + ), neutrophils (NEUT: CD45 + CD11b + Ly6G + ), DCs (CD45 + CD11c + ), and monocytes (Mono: CD45 + CD11b + Ly6C + Ly6G − ). Gating strategy is shown in C. (E) DC cell (CD11c + ) activations involving antigen presentation (SIINFEKL-MHC I and MHC II) and maturation (CD40 and CD86) at the injection site were analyzed using flow cytometry on day 3 following administration. C57BL/6 mice ( n = 6) were administered PBS, LNP, or LSE loaded with OVA mRNA via intramuscular injection (5 μg mRNA per mouse). Gating strategy is shown in D. (F) Schematic illustration (top) and flow cytometry analysis (bottom) on T cell dynamics at the injection sites. C57BL/6 mice ( n = 4) received intramuscular injections of PBS, LNP, or LSE formulations containing 5 μg OVA mRNA. Expression of CD3 + T cell CD3 + exhaustion markers (Lag-3, Tim-3, CTLA-4, and Icos) at the injection site on day 3 post-injection was assessed by flow cytometry. Gating strategy is shown in A. In (C and E), data were presented as mean ± SEM. In (F), for boxplots, the box extends from the minimum to the maximum values, with the line indicating the median. Statistical significance in (C, E, and F) was determined by one-way ANOVA with post hoc Šídák’s multiple comparisons tests. ns, p ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse: C57BL/6, female , Vital River Laboratories , N/A.

Techniques: Control, Expressing, Activation Assay, Single Cell, Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunopeptidomics

Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Potent and long-term VZV gE-specific T cell immune responses (A–F) LSE-induced enhanced VZV gE-specific T cell responses, compared with LNP. (A) gE-specific CD4 + T cell responses following prime-boost immunization of LSE and LNP (5 μg gE mRNA per C57BL/6 mouse, n = 6) with a 4-week interval. PBS served as a control. PBMCs collected at days 56, 70, 120, and 300 post-primary immunization were stimulated with gE-overlapping peptides and analyzed by ICS using flow cytometry. (B) Frequencies of gE-specific IFN-γ + (left) and IL-2 + (right) CD4 + T cells following immunization ( n = 6). (C) Cytokine secretion profiles (IL-2, IFN-γ, TNF-α, IL-21) of splenocytes at day 120 post-primary dosing, measured via ELISA assay and presented as a heatmap ( n = 6). (D) IFN-γ (top) and IL-2 (bottom) ELISpot assay of splenocytes following stimulation on day 120 after primary immunization ( n = 6). Representative ELISpot images (left) and corresponding spot-forming cell counts (right) are shown. (E) T CM (CD44 + CD62L + ) and T EM (CD44 + CD62L − ) in CD4 + and CD8 + T cells among the splenocytes at day 120. (F) Proliferation of CD4 + and CD8 + T cells at day 300 post-primary administration. T cell proliferation was evaluated using a CFSE dilution assay. Splenocytes were labeled with CFSE and stimulated with gE-overlapping peptides. After 72 h, CFSE dilution in CD4 + or CD8 + T cells was analyzed by flow cytometry to determine proliferation. (G–R) VZV gE-specific T cell response with enhanced clonal expansion and repertoire diversity, compared with Shingrix. (G) Schematic workflow of vaccination, T cell immunity assessment, and TCR sequencing. Female C57BL/6 mice ( n = 6) were immunized with LNP or LSE formulations containing 5 μg gE mRNA per mouse using a prime-boost regimen with a 4-week interval; PBS served as the control. Splenocytes collected on days 27 and 56 were sorted into T cell populations and subjected to antigen-stimulated and unstimulated TCR sequencing, and cellular immunity was analyzed at day 120. Image created with BioRender.com . (H and I) Frequencies of VZV gE-specific CD4 + (H) and CD8 + (I) T cells producing IFN-γ, IL-2, and TNF-α at day 120 post-immunization, assessed by ICS. (J) IFN-γ (left) and IL-2 (right) ELISpot assay of splenocytes following stimulation, at day 120 after immunization ( n = 6). (K and L) T CM and T EM subsets within CD4 + (K) and CD8 + (L) T cell populations among splenocytes on day 120 after primary immunization ( n = 6). (M) T cell proliferation on days 28, 56, and 120 post-primary dosing, assessed using a CFSE dilution assay. (N) Schematic of total and gE-reactive TCR repertoires analysis. Set A corresponds to total TCR repertoire from T cells sorted pre-stimulation, Set B includes gE-reactive TCR repertoire from proliferated T cells post-stimulation. Set C is intersection of two TCR repertoires. (O) Clonality of clonotypes at various stages of immunity. Bubble chart (left) shows TCR clonotypes for LSE, and Shingrix groups, with circle size indicating clonal fraction. The clonality index (right) compares the total TCR repertoire (Set A) across three groups at different immunity stages. (P) Cumulative fraction of gE-reactive clonotypes within the total TCR repertoire. (Q) Clonotype diversity of gE-reactive TCR repertoires quantified by Shannon’s index. (R) The proportion of high clonotypes (clone fraction ≥0.01%) within Set B, as a fraction of the total clonotype population. In (B, H, and I), for boxplots, the box extends from the minimum to the maximum values, with the line indicating the median. Statistical significance was determined by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (D, K, and L), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (F and M), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA compared to LSE-injected mice with post hoc Dunnett’s multiple comparisons tests. In (J), data were presented as mean ± SEM, statistically analyzed by one-way ANOVA with post hoc Tukey’s multiple comparisons tests. ns, p ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse: C57BL/6, female , Vital River Laboratories , N/A.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Dilution Assay, Labeling, Sequencing, Injection

Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Enhanced anti-tumor effects in mice (A) Schematic illustration of the vaccination regimen. Female C57BL/6 ( n = 5) mice were intramuscularly injected with LNP or LSE formulations containing 5 μg OVA mRNA per mouse. (B) Flow cytometry analysis of cell frequencies of SIINFEKL-MHCI (left), IFN-γ (middle), and granzyme B (right) T cells (CD3 + CD8 + ) in spleen ( n = 5) before and after booster immunization. (C–E) Therapeutic EG7-OVA tumor model. (C) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated subcutaneously with EG7-OVA cells and 7 days later, received a single intramuscular injection of LNP or LSE formulations containing 5 μg OVA mRNA per mouse. (D and E) Average tumor volumes (D) and survival curves (E) are shown. (F–I) Prophylactic B16-OVA tumor model. (F) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 4-week interval, followed by subcutaneous inoculation of OVA-expressing melanoma (B16-OVA) cells 4 weeks after the booster dose. (G–I) Survival curves (G), average tumor volumes (H), and represented tumor volumes (I) are shown. Mice were euthanized when the tumor volume exceeded 2,000 mm 3 . (J–M) Therapeutic B16-OVA tumor model and tumor microenvironment (TME) analysis. (J) C57BL/6 mice ( n = 6) were inoculated subcutaneously with B16-OVA cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. At 6 days post-booster immunization, tumor was extracted and T cell dynamics and cytokine secretion profiles were analyzed. (K) Average tumor volumes. (L) Expression of CD3 + T cell exhaustion markers (PD-1 and Lag-3) in the TME. (M) Cytokine levels (IFN-γ, IL-2, and TNF-α) in tumor lysates measured by ELISA and normalized by total protein content. (N and O) Metastatic B16-OVA melanoma model. (N) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated intravenously with B16-OVA cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (O) Representative lung photographs (left) and quantification of tumor nodules in the lungs (right) collected on day 28. (P–R) Tumor rechallenge model using B16-OVA melanoma. (P) Schematic illustration of the vaccination and tumor inoculation regimen. C57BL/6 mice ( n = 6) were inoculated subcutaneously with B16-OVA cells to establish the primary tumor. When the size reached 100 mm 3 , primary tumors were surgically removed; 7 days later, mice received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (Q and R) Average tumor volumes (Q) and survival curves (R). (S–Z) Prophylactic, therapeutic, and rechallenge tumor models using NY-ESO-1-highly expressing LLC. (S and T) Prophylactic LLC model. (S) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 6) received two intramuscular injections of LNP or LSE formulations containing 5 μg NYESO-1 mRNA per mouse at a 2-week interval, followed by subcutaneous inoculation of NY-ESO-1-highly expressing LLC cells 7 weeks after the booster dose. (T) Average tumor volumes are shown. (U–W) Therapeutic LLC model. (U) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 6) were inoculated subcutaneously with NY-ESO-1-highly expressing LLC cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (V and W) Average tumor volumes (V) and survival curves (W) are shown. (X–Z) Rechallenge LLC model. (X) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated subcutaneously with NY-ESO-1-highly expressing LLC cells to establish the primary tumor. When the size reached 100 mm 3 , primary tumors were surgically removed; 7 days later, mice received two intramuscular injections of LNP or LSE formulations containing 5 μg NYESO-1 mRNA per mouse at a 1-week interval. (Y and Z) Average tumor volumes (Y) and survival curves (Z) are shown. In (B, K, Q, V, and Y), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA comparing LNP-injected mice to LSE-injected mice with post hoc Šídák’s multiple comparisons tests. In (D), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (H), data were shown as mean ± SEM, statistically analyzed by two-way ANOVA compared to LSE-injected mice with post hoc Dunnett’s multiple comparisons tests. In (L and M), data were shown as mean ± SEM, statistically analyzed by one-way ANOVA with post hoc Tukey’s multiple comparisons test. ns, p ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse: C57BL/6, female , Vital River Laboratories , N/A.

Techniques: Injection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay