Figure 7. Squalene enrichment of membrane cholesterol fosters the autoreactivity of antigen-specific Th1 diabetogenic cells.

(A) Diabetogenicity of antigen (HA_110–120)-specific CD4 splenic Th1 cells was determined in RAG2 KO, RIP-HA mouse model for inducible type 1 diabetes (T1D). The HA_110–120-specific CD4 splenic T-cells (2×10⁵ cells) from either F1 mice (Foxp3-GFP^+/− TCR-HA^+/− mice) (filled circle), or from parental Foxp3-GFP⁺/⁺ mice (open circle) were infused alone, or in combination with Foxp3-GFP sorted splenic cells (2×10⁵ cells) from Fl mice (filled triangle) or Foxp3-GFP sorted cells (2×10⁵ cells) from parental Foxp3-GFP⁺/⁺ mice (open triangle) into RAG2 KO, RIP-HA Tg recipient mice (n = 5 mice/group). Shown is the mean glycemia values for individual mice in each group ± SD. Horizontal dark line indicates the upper limit of euglycemia (200 mg/dL) previously determined in a large cohort of non manipulated RAG2 KO RIP-HA Tg recipients (n = 20). Of note, only the antigen (HA_110–120)-specific CD4 splenic T-cells, but not T-regs or other non-antigen (HA_110–120)-specific T-cells were able to induce hyperglycemia in RAG2 KO, RIP-HA Tg recipients. (B) Diabetogenicity of antigen (HA_110–120)-specific CD4 splenic Th1 cells from squalene treated F1 mice was tested in the same RAG2 KO, RIP-HA mouse model for inducible T1D described in panel A. The HA_110–120-specific CD4 splenic T-cells (2×10⁴ cells) from squalene treated (180 µg/mouse) or untreated F1 mice were first depleted of Foxp3 ⁺ splenic cells 7 days post-squalene treatment, and then infused i.p. in RAG2 KO, RIP-HA Tg recipients. Shown is the cumulative incidence of hyperglycemia (X-axis) from two separate experiments, n = 5 mice/group/experiment. Cumulative incidence of hyperglycemia was calculated by dividing the number of mice per group that developed two consecutive readings of hyperglycemia (>200 mg/dL) at various intervals of time by the total number of mice and then multiplied by 100.