Fig. 6: CTA1-exposed cDC1 cells are potent inducers of Th17 differentiation.

Single cell suspensions of immunized or unimmunized mLN (mediastinal lymph node) mononuclear cells were highly enriched (>90%) for cDC1 (CD103⁺, CD11b⁻) cells by positive selection using biotinylated XCR1 Mabs at 20 h following an i.n dose of 5 μM CTA1-II-CD103. The enrichment strategy is shown with untreated cDCs from unimmunized mice and sorted cDC1 cells from mLN of immunized mice (a). Enriched migratory cDC1 cells (MHC^high, CD11c^high XCR1⁺) from unimmunized or immunized mice were incubated in triplicates for 3 days with freshly isolated OT II T cells (1:10) labeled with CFSE and the histogram shows proliferating OT II cells in cultures with cDC1 from immunized or unimmunized mice and values are given as mean % ±SD of pooled cDC1 cells from 3–5 mice in each group and two independent experiments (b). The frequencies are given as mean % ±SD of Th17 (rorγt⁺) and Th1 (Tbet⁺) CD4 T cells on day 3 in these cultures of proliferating OT II cells with unimmunized as opposed to immunized cDC1 cells (c). Cytokine production, as indicated, in these cultures with unimmunized or immunized cDC1 cells is given in pg/ml ±SD for each group (d). In vitro triplicates cultures with enriched cDC1 cells from unimmunized mice were co-cultured with naïve Eα-peptide-specific TCR Tg CD4 T cells (1:10) and 1 μM Eα-peptide together with or without different adjuvants added simultaneously; CTA1-II-CD103 (1 μM), Poly I:C (10 μg/ml), LPS (10 μg/ml), CpG (2.5 μg/ml) or unstimulated (\), as indicated. The frequency of Rorγt⁺ or Tbet⁺ Eα-specific TCR Tg CD4 T cells in cultures was determined on day 3 and the frequency of intracellular IFNγ⁺ cells was assessed by flow cytometry using labeled specific antibodies. Two independent experiments (I and II) are shown giving similar results (e). Statistical significance was calculated using ANOVA with Dunnett’s posttest and p values are given as; ****p < 0.0001.