Figure 7. Intermittent 3HB scheduling affects systemic expression of T cell inhibitory receptor and their ligands.

(A and B) Experimental setting for intemittent diet and combination immune checkpoint blockers (cICB) in the RET model and Kaplan Meier curves of overall survival for 12 animals/group, according to therapeutic scheme comparing continuous (Cont) versus intermittent (On/off) diet interventions. (C) Flow cytometric analyses of PD-1 and CTLA-4 expression in splenic CD8⁺ T lymphocytes at day 17, after diet (ketogenic diet [KD] or 3-hydroxybutyrate [3HB] Cont or On/off) and systemic cICB combinatorial regimen in RENCA bearing BALB/c mice. Refer to Supplemental Figure 5, A and B, for Tim-3, Lag-3, and 4-1BB in all splenic T cells. (D and E) Flow cytometry analysis of MFI for the membrane expression of costimulatory (CD86 in D) or inhibitory (PD-L1 in E) on macrophages (D) and other myeloid subsets from the spleens at day 17, after complete diet (3HB On/off) and systemic cICB combinatorial regimen in RENCA-bearing BALB/c mice. The results from 2–3 experiments involving 6 mice/group are depicted; each dot represents 1 spleen. (F and G) In vitro effects of increasing dosing of 3HB onto a DC cell line stimulated or not with rIFN-γ. Flow cytometry determination of MHC II/I-Ab (F) and PD-L1 expression (G) as mean fluorescence intensity (MFI) on the surface expression at 48 hours of stimulation. One representative experiment out of 4 is depicted, yielding similar conclusions (n = 12 or each groups). Student’s t test (C), global comparison using Kruskall-Wallis test with post hoc multiple comparisons using Dunn’s test (*P < 0.05) (D and E). Global comparison using ANOVA with post hoc multiple comparisons using Dunnett’s test (*P < 0.05) (F and G). *P < 0.05, **P < 0.01, ***P < 0.001.