Fig 2: Selective IDO1 gene deletion in cDC1 worsens EAE(A) EAE score in WT and Irf8 Δ32−/− mice. Five mice per group, n = 3.(B) IL-17, GM-CFS, and TGF-β in supernatant of cervical LNs from EAE mice in (A) at day 28 and restimulated in vitro with MOG 20 μg/mL for 5 days.(C) H&E staining of spinal cord sections from EAE mice in (A).(D) Flow cytometric analysis of FoxP3+CD25+ cells in cervical LNs of mice immunized as in (A).(E) Schematic representation of Ido1f/fXcr1Cre/+ strain generation.(F) IDO1 immunoblot. Left: cDC1 untreated or treated with LPS 250 ng/mL for 48 h. Right: macrophages (MΦ) untreated or treated with LPS 250 ng/mL and IFN-γ 15 ng/mL for 48 h β-actin was used as loading control (n = 3).(G) Flow cytometric analysis of PDL1+ in thymic cDC1.(H) EAE score in Ido1f/f Xcr1+/+ and Ido1f/fXcr1cre/+ mice. 9 mice per group, n = 2.(I) H&E staining of spinal cord sections from mice treated in (H).(J) Quantification of cDC1 and cDC2 in spinal cords from EAE mice in (H) at day 26.(K) PDL1+ cDC1 frequency out cDC1 by flow cytometry in spinal cords of EAE mice in (H).(L) Cytokine analysis in supernatants from cervical LNs of EAE mice in (H) at day 26 stimulated as in (B).(M) Flow cytometric analysis of FoxP3+CD25+ cells in cervical LNs of mice immunized as in (H).(N) Tryptophan metabolites in plasma from EAE mice in (H) at day 26.(O) Il6 mRNA expression in cervical LNs from EAE mice in (H).(P) EAE score in Irf8 Δ32−/− mice transferred with cDC1 genotypes at days 4 and 7 post immunization. 5 mice per group, n = 3.Data are mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA followed by Bonferroni multiple comparison test (A, H, and P) or unpaired t test (B, D, G, and J–O). Each dot represents an individual value. Please also see Figure S3.

Fig 3: The Th17 Response to Bph Is Antigen Specific and Aids Controlling Colonization(A) Left panel: Concatenated flow cytometry plots of cultures of splenocytes of five mice taken 52 days after inoculation with either HK or live Bph. Splenocytes were loaded with either no protein or Bph proteins from a HK culture. Cells were gated on CD4+TCRß+ cells using CD25 as an activation marker and CFSE as a proliferation marker. Representative of 2 independent experiments. Right panel: Quantification of CD25+CFSElo T cells from splenocyte cultures as shown in (A). n = 9–10 mice/group, combined from 2 independent experiments.(B) IL-17A ELISA of culture supernatants from lung CD4+ T cells co-cultured with antigen-loaded CD11c+ dendritic cells (DCs). CD4+ T cells were isolated from the lungs of five mice taken 43 days after inoculation with either HK or live Bph.(C) CFU of Bph recovered from BALs of WT or RAG1-/- mice 14 days after colonization. n = 10 mice/group, combined from 2 independent experiments.(D) Top: Schematic of the experimental approach to test the role of IL-17A during Bph colonization. Bottom: CFU of Bph recovered from lung homogenates (CFU/g), BAL, or nasal lavage (CFU/ml).(E) ELISA showing SLPI protein expression in the lungs of mice inoculated with Bph and treated with anti-IL-17A monoclonal antibody compared to the isotype control.Statistical significance: Kruskal-Wallis followed by post hoc one-tailed paired Wilcoxon rank-sum test with adjustment for multiple hypotheses using BH correction for (A) and (B) or two-tailed Wilcoxon rank-sum test for (C)–(E). Horizontal lines indicate median values. **p < 0.01; ***p < 0.001.

Fig 4: Tick EVs regulate DETCs during hematophagy.I. scapularis scV33 (blue) or siV33 (red) ticks were placed on C57BL/6 mice and allowed to feed for 3 days. On day 3, biopsies were taken from the skin at the bite site and compared to the naïve treatment (gray). (A) DETC (Vγ5), (C) JAML, (D) NKG2D, (E) CD69, (F) CD25 and (H) CD100 cells were assessed by flow cytometry. Graphs represent 1 of 3 independent experiments. (B) Schematic representation of the DETC-keratinocyte crosstalk at the skin epidermis. (G) Epidermis containing Langerhans cells (red), DETCs (green), and keratinocytes (white) imaged on day 3 after injection with phosphate buffered saline (PBS - mock) or EV (4×107 particles) into the mouse ear. Nocodazole (100 μg/ml) was applied topically on the mouse ear to induce DETC rounding as a positive control. Langerhans cells, DETCs and epithelial cells were simultaneously visualized in the huLangerin-CreER; Rosa-stop-tdTomato; CX3CR1-GFP+/−; K14-H2B-Cerulean mouse strain. Cre expression was induced with an intraperitoneal injection of tamoxifen (2 mg). Statistical significance shown as *p<0.05, ns = not significant. Data are presented as a mean with standard deviation. Significance was measured by One-way ANOVA followed by Tukey’s post hoc test.

Fig 5: Tick EVs impact keratinocyte proliferation.(A) Ingenuity pathway analysis derived from siV33 compared to the bite of scV33 ticks on FVB-Jax or FVB-Tac mice. Canonical pathways predicted to be inhibited (blue, negative z-score) or activated (orange, positive z-score) based on differential expression profile. The solid line indicates the p-value significance threshold of 0.05 (−log=1.3). (B) The signaling cascade of EIF2 (highlighted in yellow, A), yielding (→) or inhibitory (┤) arrows. Orange indicates activation whereas blue shows inhibition according to the IPA prediction. Gene expression based on the scRNAseq experiment is indicated in red (increased) or green (decreased). Gray – no expression or prediction. (C) ScV33 (circle) or siV33 (square) injected I. scapularis nymphs were fed on FVB-Jax (white) or FVB-Tac (gray) mice for 3 days. Biopsies were taken from the skin at the bite site and assessed for EpCAM+ Ki67+ keratinocytes by flow cytometry. (D-I) ScV33 or siV33 ticks fed on C57BL/6 mice for 3 days. Biopsies were taken from the skin at the bite site and processed for flow cytometry analysis. (D) PI3K p85+, and (E) phospho-PI3K p85/p55+. (F) ELISA analysis of KGF normalized to total protein per 5 mm skin punch biopsy. (G) EpCAM+ Ki67+ keratinocytes assessed by flow cytometry. Graph displays proliferation changes within the scV33 or siV33 treatments compared to the naïve skin. Data are presented as a mean with standard deviation. Statistical significance shown as *p<0.05, ns = not significant. (C-G) Significance was measured by One-way ANOVA followed by Tukey’s post hoc test. (H-I) Flow cytometry histogram plots of EpCAM+ Ki67+ keratinocytes. (H) scV33 or (I) siV33 treatments displayed according to the number of ticks bitten per biopsy. X-axis shows fluorescence intensity, and the Y-axis indicates the count of events in the fluorescence channel.