Extended Data Fig. 9: Ox-mtDNA enables primary pDC to induce Tfh differentiation by activating NLRP3.
a, Naïve B6 splenic CD4+ T cells were co-cultured with purified peritoneal pDC collected 24 h after 4 repetitive mtDNA or Ox-mtDNA injections (72 h apart). No peritoneal pDC were present after PBS injection. After 3 days of co-culture, Tfh (CXCR5+BCL6+Foxp3-CD44+CD4+) generation was analyzed (n = 3 independent experiments). b, BM Flt3L-induced BMDC (CMFDA+) were i.p. injected into 8-week-old B6 males pre-treated with 4 repetitive PBS or mtDNA injections. 24 h later, splenic DC were analyzed for presence of peritoneal CMFDA+ pDC (n = 3 independent experiments). c, Naïve B6 splenic CD4+ T cells were co-cultured with purified splenic DC collected 24 h after 4 repetitive PBS or Ox-mtDNA injections into DT treated BDCA2-DTR- and BDCA2-DTR+ mice. After 3 days, Tfh generation was analyzed (n = 5 independent experiments). d, Experimental strategy for generating mixed BM chimeras by transplanting lethally irradiated females with 50:50% mixtures of Nlrp3−/− and BDCA2-DTR− (bearing Nlrp3+/+) or BDCA2-DTR+ (bearing Nlrp3+/+) BM, followed by i.p. injections of DT and Ox-mtDNA, as indicated. e-h, Serum titers of anti-dsDNA and anti-nucleosome IgGs (e), percentages of splenic GC, IgG1+IgM-CD19+, IgG2b+IgM-CD19+ B cells and Tfh (f). serum IL-21 amounts (g) and spleen mass to body weight ratio (h) (n = 4/group). Results (a-c and e-h) are mean ± SD. Kruskal–Wallis test (a, b) and two-way ANOVA with Tukey multiple-comparison test (c and e-h).
