Figure 6

TNFR2 mediates Fas upregulation, and blocking TNFR2 inhibits Fas upregulation in Idd9 congenic but not NOD islets. (A) Islet Fas expression is induced only by combined TNF+IFNγ treatment. Intact islets from B6 mice were treated with medium, 1,000 U/ml IFNγ and 1,000 U/ml TNF, alone and in combination, for 48 hours. Islets were then dispersed into single cells and, after 60 min recovery incubation, stained with antibodies to Fas or the relevant isotype control for anlysis by flow cytometry. The average %Fas+ cells for each treatment, gated on live FL1+ β cells, is shown. (B) TNFR2 mediates Fas upregulation in β cells. Islets from B6 and B6.TNFR2KO mice (pooled from 2 mice for each strain) were treated in triplicate with medium or IFNγ +TNF (both at 1,000 U/ml) for 48 hours and stained with antibodies to Fas as in (A). Control samples were stained with the relevant isotype control. The % Fas+ cells +/- SEM, gated on live FL1+ β cells, is shown in (C). Representative of 4 independent experiments. (D) Blocking TNFR2 inhibits the upregulation of Fas in β cells from Idd9 congenic but not NOD mice. Islets from 5 week old Idd9 congenic and NOD mice were treated as before, except that IFNγ +TNF treated islets were pre-incubated for 60 min with 2 μg/ml blocking anti-TNFR2 antibody (I+T+R2), or the relevant isotype control (I+T). Representative dot plots are shown and values indicate the average %Fas+ cells (isotype corrected), gated on live FL1+ β cells, and this data +/- SEM is plotted in (E). The data was pooled from 6 separate experiments with total n = 13–14.