FIGURE 3.

Granule cytolytic granzyme C (Gzm C) in CD8αα⁺RT1B/D⁺Ly49s⁺ cells mediates killing of autoreactive effector T cells. (A) RT-PCR detection of granule cytolytic proteins in various cell populations. G, glomeruli-infiltrating; P, PBMC; Gzm, granzyme; Pfr, perforin. (B and C) Combination of immunofluorescence on RT1B (green) and ISH with full length gzmc antisense RNA (anti-s) as a probe (red) demonstrate gzmc mRNA in PBMC CD8αα⁺RT1B/D⁺Ly49s6⁺ cells; cells probed with sense RNA were negative. In (B), one double negative and one single RT1B/D⁺ cell (green) are shown as internal controls. Bars=3 μm. (D) Immunofluorescence/ISH shows gzmc mRNA in CD8αα⁺RT1B/D⁺Ly49⁺ cells (arrows) in glomeruli (outlined by dash line) of an immunized rat. Bar=30μm. (E) Immunofluorescence shows cytoplasmic Gzm C (red) in a glomeruli-infiltrating CD8αα⁺RT1B/D⁺Ly49s⁺ cell (green); two nearby double negative T cells are shown as internal controls. Bar = 3 μm. (F) Western Blot on the proteins from various cells shows Gzm C of 56kD in CD8αα⁺RT1B/D⁺Ly49s⁺ cells but not in T cells. (G) Killing assay under various conditions as indicated shows reduced killing efficacy of target T cells in the presence of anti-Gzm C Ab (α-Gzm C). Glomeruli-infiltrating CD8αα⁺RT1B/D⁺Ly49s⁺ cells (G CD8αα⁺) were used as killing effectors and pCol(28–41)-specific T cells as targets. Killing was expressed as % of CFSE-labeled target T cells in control wells without killing effectors. Three independent experiments were conducted with similar results. One-way ANOVA with Tukey post test was performed. *, p<0.05, **, p<0.01, ***, p<0.001.