FIGURE 4.

CD8αα⁺RT1B/D⁺Ly49s6⁺ cells express intracellular Fas-L. (A) qPCR array for apoptosis pathway on glomerular mRNAs at early inflammation (day 20 post immunization) and T cell apoptosis peak (day 35) of immunized WKY rats; apoptosis-inducing receptors and ligands are shown separately and paired; expression of each gene is expressed as fold changes between day 20 and day 35; note that significant increases are seen in both Faslg and Ltα. (B) RT-PCR shows the detection of a high level of Faslg mRNA in the isolated glomeruli-infiltrating CD8αα⁺RT1B/D⁺Ly49s⁺ cells (CD8⁺RT1B⁺), but no Ltα expression was detected. (C) Two color flow cytometry on isolated glomeruli-infiltrating CD8αα⁺RT1B/D⁺Ly49s6⁺ cells for the detection of Fas-L; permeabilized cells showed a Fas-L⁺CD8α⁺ population (gated). (D) Immunofluorescence shows intracellular Fas-L (green) in a permeabilized CD8αα⁺RT1B/D⁺ cell (red) (right panel), but not in a live one (left panel). Bar =3 μm. (E) Killing assay shows that addition of Fas-L Ab (α-Fas-L) did not inhibit CD8αα⁺RT1B/D⁺Ly49s6⁺ cell-mediated killing of pCol(28–40) specific T cells. Three independent experiments were conducted with similar results. One-way ANOVA with Tukey post test was conducted. ***, p<0.001.