Extended Data Fig. 3: mCSF1R CART do not persist in immunocompetent mice.

(a) Construct design of mCSF1R or mEpCAM CART or mCherry T cells. (b) Representative histograms of mCSF1R or mEpCAM expression on J774A.1 cells. Staining was carried out twice. (c) Summary of treatment schedule for in vivo toxicity assessment of mCSF1R CART. (d) Mean weight curves of mice treated with 3 × 10⁶ mCSF1R CART or mCherry T cells. n = 10 mice per group. Error bars indicate s.e.m. (e) Quantification of tissue-resident CD11b + cells (left) or mCherry+ T cells of parent population (right, parent population: CD3 and CD8 positive cells) in different organs by flow cytometry. Data are mean ± s.e.m. of n = 10 mice. (d, e) Statistical significance was calculated using two-way ANOVA with Šidák multiple comparison correction. (f) Scheme of treatment schedule for in vivo toxicity assessment of mCSF1R CART. WBI, whole body irradiation. Mice were treated with 3 × 10⁶ mCSF1R CART or mCherry T cells per mouse. 6 × 10⁶ mEpCAM CART were transferred as a positive control. (g) Serum levels of indicated markers one (d1) or seven (d7) days after ACT. Depicted is the fold change of serum levels of the indicated groups from the PBS-treated control group. CRP, C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GLDH, glutamate dehydrogenase; γ-GT, gamma-glutamyl-transferase; LDH, lactate dehydrogenase. n = 3 mice per group. Statistically significant increases in the serum of mEpCAM CART treated mice compared to mCSF1R CART or control-treated mice at day 7 were observed for GLDH (p < 0.0001). (h) Histopathological analysis of indicated organs after treatment with mEpCAM or mCSF1R CART or mCherry T cells. Representative images of n = 3 mice per group. Signs of organ damage are indicated in the picture: black arrowhead: thickening of alveolar epithel. (i) Representative maximum intensity projection of microglia (green) and macrophages (green) in CX3CR1-GFP mice on days 0 (baseline), 4, 7, 10, 14, 21 and 28 after intravenous injection of 10⁷ mCSF1R CART (red). Depth from brain surface: 0–100 µm. Scale bars as depicted: 50 µm and 20 µm. mCSF1R CART: i.c. injection, n = 5 mice; i.v. injection, n = 3 mice; mCherry T cells: i.c./ i.v. injection = 2 mice.