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[Preprint]. 2023 May 3:2023.04.28.538637. [Version 2] doi: 10.1101/2023.04.28.538637

Figure 3: M-CSF secreting “Self-Sustaining” Aβ CAR-Ms have improved survival and reduce plaque load in vivo.

Figure 3:

a) Schematic of M-CSF secreting “self-sustaining” construct retrovirally introduced into control and Aβ CAR-Ms that contains the M-CSF gene followed by a P2A cleavage sequence and Thy1.1. b) % change in live cell count of first-generation Aβ CAR-Ms and self-sustaining Aβ CAR-Ms upon removal of M-CSF from the culture medium in vitro, determined by flow cytometry staining with ZombieNIR live/dead staining. Cells were differentiated for 6 days in M-CSF to become mature macrophages, prior to M-CSF removal. Statistical significance was calculated with an unpaired t-test. c) Schematic of PLX5622 pre-conditioning and intrahippocampal injection of self-sustaining Aβ CAR-Ms. d) Total flux determined by non-invasive bioluminescence imaging (BLI) tracking first-generation Aβ CAR-M kinetics in vivo compared to self-sustaining Aβ CAR-M kinetics after intrahippocampal injection (upper). Representative BLI images from self-sustaining Aβ CAR-M treated mice (lower). Days indicates days post-intrahippocampal injection. n=10–18 mice per group. Statistical significance was calculated with unpaired t-tests. e) Fold-expansion of CAR-Ms from the first day of BLI after intrahippocampal injection of cells to the day of maximum total flux measured by BLI. n=6–18 mice per group. Statistical significance was calculated with one-way ANOVA with Tukey’s multiple comparisons test. f) Representative immunofluorescence microscopy images of self-sustaining Aβ CAR-Ms binding to amyloid plaque in vivo. g) Assessment of plaque load after intrahippocampal injection of self-sustaining control CAR-M or self-sustaining Aβ CAR-M in n=7 aged APP/PS1 mice. Mice were sacrificed on day 12 or 13 post intrahippocampal injection and brain tissue was sectioned and stained with HJ3.4 and X-34 to assess plaque load. Data shown as mean ± s.e.m. Statistical significance was calculated with unpaired t-tests. For b-d, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.