Fig. 5. Efficient T-cell targeting in immunocompetent mice.

B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai reporter) mice were injected intravenously with three daily doses of nanoparticles loaded with 15 μg mRNA encoding nuclear localization signal (NLS)-Cre. Nanoparticles were targeted to mouse T cells using a full-length anti-CD3 MuIgG2a, or IgG2a isotype control. Both antibodies were designed as LALAPG variants to ablate Fc receptor binding and complement activation. Forty-eight hours after the final injection, organs were collected and whole-organ dtTomato fluorescence was measured using fluorescent IVIS imaging. Single cell suspensions of spleens and blood were labeled with antibodies against various immune cell subtypes and analyzed by flow cytometry. a Representative (N = 7, 1 pictured) dtTomato expression in organs under fluorescent IVIS imaging. b Quantification of fluorescent signal in each organ. Each symbol indicates one measured organ. Horizontal lines indicate mean values, and error bars represent standard deviation of the mean. Pairwise differences in fluorescent intensities between the groups were statistically analyzed by two tailed unpaired Student’s t-test. N = 7 biologically independent samples pooled from two independent experiments. c Graph displaying the mean ± SD percent of immune CD45+ dtTomato+ cell types in the spleen. Macrophages (CD45+, CD11b+, MHCII+, CD11c−, Ly6C−/low, Ly6G−), B cells (CD45+, B220+), T cells [CD4+ T cells (CD45+, TCR-β chain+, CD4+, CD8-), CD8+ T cells (CD45+, TCR-β chain+, CD4−, CD8+)], neutrophils (CD45+, CD11b+, MHCII+, CD11c−, Ly6G+), and dendritic cells (CD45+, CD11c+, CD11b−, MHCII+) were measured. Each symbol indicates one mouse. Horizontal lines indicate mean values, and error bars represent standard deviation of the mean. Pairwise differences in fluorescent intensities between the groups were statistically analyzed by two tailed unpaired Student’s t-test. N = 7 biologically independent samples.