Figure 7: IL-17-producing Il1rn^−/− Vγ6⁺ γδ T cells intrinsically express high levels of IL-1R.

(a) Flow cytometry of joint-infiltrating cells from Il1rn^−/− mice (n=3) for the expression of CCR2 and Vγ4. Numbers refer to percentage in CD3ɛ⁺ γδ TCR⁺ cells. Numbers in parentheses represent percentage in Vγ6⁺ (Vγ4⁻) or Vγ4⁺ cells (left). Quantification of CCR2⁺ cells in Vγ6⁺ (Vγ4⁻) or Vγ4⁺ cells is shown (right). NS, not significant versus Vγ4⁺ cells (unpaired Student’s t-test). (b) Flow cytometry of IL-1R expression in LNs and joint-infiltrating cells from WT or Il1rn^−/− mice. Numbers refer to percentages in CD3ɛ⁺ cells. Numbers in parenthesis represent percentages in CD3ɛ⁺ γδ TCR⁺ cells (left). The percentage of IL-1R⁺ cells among CD3ɛ⁺ γδ TCR⁺ cells is indicated on the right. **P<0.01; ***P<0.001 (versus WT mice) (unpaired Student’s t-test). (c) FACS analysis of IL-1R and GFP expression on joint-infiltrating CD3ɛ⁺ γδ TCR⁺ cells from WT, Il1rn^−/− and Il17^g/g Il1rn^−/− mice. Numbers in parentheses represent percentage in GFP⁺ γδ TCR⁺ cells. (d,e) Flow cytometry of IL-1R expression in joint-infiltrating cells from Il1rn^−/− mice. Numbers refer to percentages in CD3ɛ⁺ γδ TCR⁺ cells. Numbers in parentheses represent percentages in Vγ6⁺ (Vγ4⁻) or Vγ4⁺ cells (d, left). Quantification of IL-1R⁺ cells (d, right) and mean fluorescence intensity of IL-1R in Vγ6⁺ or Vγ4⁺ cells are indicated (e). *P<0.05; ***P<0.001 (versus Vγ4⁺cells) (unpaired Student’s t-test). (f–h) Flow cytometry of IL-1R expression in γδ T cells in newborn thymus (within the first day after birth) from WT and Il1rn^−/− mice. IL-1R expression on γδ T cells (f) and on different Vγ subsets (g), and CCR2 and IL-1R expression on γδ T cells (h) are shown. Numbers refer to percentage in CD3ɛ⁺ cells (f,h, top), or in CD3ɛ⁺ γδ TCR⁺ cells (g,h, bottom). Numbers in parentheses represent percentage in γδ TCR⁺ cells (f,h, top) or in IL-1R⁺ γδ TCR⁺ cells (h, bottom). (i,j) qPCR analysis of the transcripts for IL-1β (Il1b) (i) or IL-23 (Il23p19) (j) in cells from joints, draining LNs (dLN), mesenteric LNs (mLN) and spleens (SP) from WT or Il1rn^−/− mice. mRNA levels were normalized against Gapdh, and values are shown relative to mLN of WT mice. *P<0.05; NS, not significant (versus WT mice) (unpaired Student’s t-test). Data show mean±s.e.m. All data are representative of two or three independent experiments.