Extended Data Fig. 6: Ox-mtDNA treatment of mouse and human pDC enables ex vivo induction of Tfh differentiation.

a, Naïve splenic CD4⁺ T cells (see gating strategy in Supplementary Fig. 4a) were co-cultured with purified peritoneal or splenic pDC collected 24 h after two alum injections (72 h apart). Tfh generation was analyzed 72 h after (n = 5). Created with BioRender.com. b, BM Flt3L-induced BMDC labeled with CellTracker Green CMFDA were i.p. injected into B6 males. After 24 h, the presence of peritoneal CMFDA⁺ pDC in spleen was examined (n = 3 biological replicates). c, Naïve CD4⁺ T cells were co-cultured with FACS-sorted BM Flt3L-induced pDC (see gating strategy in Supplementary Fig. 4b), −/+ mtDNA or Ox-mtDNA (50 μg) for 72 h. Tfh percentages were analyzed (n = 5). d,e, Tfh percentages after naïve CD4⁺ T cells co-culture with either pDC, cDC1, or cDC2, −/+ Ox-mtDNA (d) or with pDC incubated with different types of DNA (e) (n = 3). f, Naïve CD4⁺ T cells were cultured in transwell plates separately from or together with pDC incubated with Ox-mtDNA for 72 h. Tfh differentiation in either the upper (left) or lower (right) wells was analyzed (n = 4). g, Treatment and analysis scheme for h,i. Created with BioRender.com. CD4⁺ T after naïve CD4⁺ T cells co-cultured with pDC, −/+ mtDNA or Ox-mtDNA were isolated and co-cultured with naïve splenic B cells for 72 h, after which B cell proliferation, differentiation and IgG secretion were analyzed. h,i, Percentages of different B cell types (h) and ELISA of secreted IgG (i) (n = 4). j,k, Secreted IgG (j) and BCL6⁺ B cells and plasmablasts (PB; CD138^hiB220^loIgD^-CD3^-CD19⁺, k) 72 h after co-culture of naïve splenic B cells with CD4⁺ T cells from PBS or alum injected mice (n = 4). l,m, Freshly isolated pDC and CD4⁺ T cells purified from healthy human buffy coats were co-cultured as in c. Tfh percentages (l) and intracellular BCL6 (m) were analyzed (n = 3 biological replicates). Results are mean ± SD. Kruskal–Wallis test (c, f and h-l) and two-way ANOVA with Tukey multiple-comparison test (a, d and e).

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