Fig 2: Intranasal delivery of rAAV2-IL-15-infected microglion-shaped M/MF phenotype. (A) Quantification of Iba1+, Arg1+, and Iba1+/Arg1+ cells in the tumor area upon i.n. delivery of rAAV2-GFP or rAAV2-IL-15 microglia. Bars represent the mean (± s.e.m.) area expressed as percentage of total tumor areas. Representative immunofluorescence images are shown on the right (N = 4 mice per condition; **p < 0.01, *p < 0.05 Student’s t test). Scale bar: 20 µm. (B) Quantification of area of the branching, soma, and scanning domain of Iba+ cells measured by ImageJ in slices from GL261-bearing mice treated, as indicated (six slices, four mice per condition, **p = 0.0034, Student’s t-test). Below: representative images of maximum intensity projections of a confocal z-stack imaging on peritumoral area of Iba+ cells, converted to binary images and then skeletonized by the Analyze Skeleton plugin in ImageJ (green lines).

Fig 3: rAAV2-IL-15-infected microglia increase NK cell viability. (A) RT-PCR of the il-15 gene in primary microglia obtained from pups (P0–2) incubated for 7 days with vehicle, rAAV2-GFP, and rAAV2-IL-15 at different dilutions (n = 3, data are the mean ± s.e.m, **p < 0.01 one-way ANOVA). (B) Colocalization of IL-15- and Iba1-covered areas in primary culture of microglia treated with vehicle, rAAV2-GFP, and rAAV2-IL-15 (1:9) for 7 days (n = 6 data are the mean ± s.e.m. **p < 0.01 one-way ANOVA). For boxplots, the center line, boxes, and whiskers represent the median, inner quartiles, and the rest of the data distribution, respectively. Right: representative immunofluorescence of primary cultured microglia (stained with Iba1 in green) and IL-15 (red). Scale bar: 20 µm. (C, D) After 7 days of incubation with rAAV2-GFP and rAAV2-IL-15 (1:9), primary cultures of microglia were incubated with NK cells, isolated from the spleen of C57BL/6 mice, in a 1:1 ratio, in the presence of IL-15 (50 ng/ml), a specific Ab against IL-15 or vehicle; NK cell number (C) and CD69+ and NKG2D+ NK cells (D) were assessed by FACS analyses (n = 6, *p < 0.05 one-way ANOVA). Error bars show mean ± s.e.m.