Fig. 2: In vivo assessments of local microenvironment generated by LiNx.

a Recruitment of host cells was assessed two weeks after s.c. administration of three different LNP/mRNA LiNx formulations in C57BL/6 mice (30 μg mOVA per mouse). The Cellaca MX High-throughput Automated Cell Counter was employed to count the living cells within the composites. b–d On day 14, flow cytometry was utilised to determine the counts of CD3⁺CD4⁺ T cells (b), CD3⁺CD8⁺ T cells (c), and CD11b^–CD19⁺ B cells (d) within the composite. e Composition of host immune cells recruited within the composite was evaluated at two weeks after s.c. administering three different LiNx formulations in C57BL/6 mice (30 μg mOVA per mouse). This analysis included CD4 T cells, CD8 T cells, B cells, DC-like cells (CD11b⁺ CD11c⁺), macrophage-like cells (CD11b⁺CD11c^–), neutrophils (CD11b⁺Ly6G⁺), and NK cells (CD11b^–NKp46⁺). f–g, RT-PCR array analysis was conducted using RNA isolated from the local microenvironment generated by C10, D6, and F5 LiNx formulations. C57BL/6 mice received the three different LiNx formulations loaded with mOVA through s.c. injection (30 μg mOVA per injection). The presented heatmap shows a panel of genes relevant to immune responses (f). Subsequent RT-PCR analysis of selected genes from the panel confirmed that D6 LiNx promoted critical proinflammatory cytokine transcripts involved in the establishment of a local immunostimulatory niche. These included genes related to inflammatory cytokines and chemokines (IL-6, IL-1α, CSF-3, and CXCL10), Th1 response (TBX21, TNF, IFN-γ, GZMB, NOS-2, IL-12α, IL-15, and IL-2), and genes associated with Th2 response (IL-4, CCR-4, and IL-13). Data represent mean  ±  s.e.m. (n =   7 mice for a–e and n = 3 mice for f–g). Data were analysed using one-way ANOVA and Tukey’s multiple comparisons test for a–d. *P < 0.05. Source data are provided as a Source Data file.