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. 2024 May 9;134(12):e179848. doi: 10.1172/JCI179848

Figure 2. Maternal humoral immunity to Cas9 endonuclease is transferred to the fetus, while maternal T cell immunity to Cas9 endonuclease is not.

Figure 2

(A) Anti-SpCas9 IgG BAb ELISA. Serum concentrations of anti-SpCas9 IgG BAb were compared among the groups 28 days after primary sensitization using 1-way ANOVA. (B) Maternal-fetal transmission of anti-SpCas9 IgG BAb. Shown are the absolute and relative concentrations of anti-SpCas9 IgG BAb among three SpCas9-sensitized dams and their respective newborn fetuses. The mean fetal/maternal ratio for each pregnancy was compared by 1-way ANOVA. (C) Anti-Cas9 delayed-type hypersensitivity (DTH) reaction gross histology. Shown are images of the bilateral ears 48 hours after intradermal injection with Cas9 protein. Red arrows point to erythema and swelling consistent with a positive DTH reaction. (D) Ear skin microscopic histology. Shown are cross-sectional H&E-stained slides of the ears 72 hours after intradermal injection imaged at ×10 original magnification with ×25 original magnification inset images. Green arrows point to infiltration with small blue lymphocytes and associated edema consistent with a positive DTH reaction. Scale bars: 200 μm. (E) Measurement of ear thickness. Ear thickness was measured using microcalipers at 24, 48, and 72 hours after injection and compared among the groups using 2-way ANOVA. Significantly increased thickness was observed in the ears of SpCas9-sensitized and SaCas9-sensitized dams injected with the same Cas9 subtype to which they had been sensitized. (F–H) Anti-Cas9 DTH reaction among offspring. Unsensitized, SpCas9-sensitized, and SaCas9-sensitized BALB/c dams were mated with mTmG+/+ males and allowed to deliver. Pups were fostered and tested at 6 weeks of age. Gross histology (F), microscopic histology (G), and change in ear thickness compared by 2-way ANOVA (H) all demonstrated a negative anti-Cas9 DTH reaction, showing that maternal T cell immunity was not efficiently transferred from dam to offspring. Scale bars: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.