Fig 2: In vitro differentiation of CRISPR-modified BM cells into macrophages and dendritic cells. (A) Model describing the experimental setup. (B) Flow cytometry plots showing the gating strategy for cells differentiated for seven days in M−CSF. GFP KO percentage (C) and InDel frequency (D) in sorted macrophages (viable, F4/80+, CD11b + singlets) from M−CSF cultures. (E) Macrophage differentiation efficiency in Lin- Cas9 + cells electroporated +/- GFP sgRNA. (F) Macrophages generated from control and GFP sgRNA electroporated Lin- Cas9 + BM cells were mixed 1:1, incubated with PE/IgG phagocytosis beads, and 30 mins later analyzed for binding efficiencies. Representative FACS plots shows gating for GFP + and GFP- viable singlets, followed by analysis of F4/80 and CD11b expression. (G) Quantification of phagocytosis efficiency (% PE + ) in GFP- and GFP + macrophages. (H) Flow cytometry plots showing the gating strategy for Lin- Cas9 + cells electroporated +/- GFP sgRNA and then differentiated for nine days in Flt3L. (I) Frequency of GFP KO by flow cytometry in dendritic cells (viable, CD11c+, MHC II + [I-A/I-E + ] singlets) from Flt3L cultures. (J) Dendritic cell differentiation efficiency in Lin- Cas9 + cells electroporated +/- GFP sgRNA. Data shown as representative flow cytometry plots (B, F, H), mean and individual data (C, D, E, G, I, J, n = 3). n.s. = non-significant, *** = p < 0.001 by unpaired T-test (C, D, E, G, I, J).

Fig 3: cDC1 abundance in the medulla is locally sensed by preDCs and immature cDCs via Flt3L availability(A) Frequency of CD135+ cells among CD11c+MHCII− cells that contain preDCs in popliteal LNs of WT mice 16, 24, and 40 h after s.c. MVA infection into the foot hock.(B and C) Surface CD135 expression among preDCs (CD11c+MHCII−) (B) and immature (MHCIIloCD24+, left) or migratory (XCR1+CD8α−CD103+, right) cDC1s (C) in draining LNs of Zbtb46GFP mice 24 h after s.c. IFNα injection into the foot hock.(D and E) Surface CD135 expression among preDCs (CD11c+MHCII−) (D) and immature (MHCIIloCD24+, left) or migratory (XCR1+CD8α−CD103+, right) cDC1s (E) in draining LNs of Zbtb46GFP mice 16 h after i.p. Flt3L injection.(F) Surface CD135 expression among preDCs (CD11c+MHCII−) (left) and immature (MHCIIloCD24+, right) cDC1s in draining LNs of WT mice 24 h after s.c. IFNα injection into the foot hock and 8 h after Flt3 inhibitor administration.(G) Frequency of CD135+ cells among CD11c+MHCII− cells that contain preDCs in LNs of Xcr1DTR mice 2, 3, and 4 days after cDC1 depletion with DTx injection.(H and I) Mixed Xcr1DTR:Xcr1Venus (50:50) BM chimeras were treated with DTx 2 days before analysis to deplete only half of the cDC1 population. Frequency of CD135+ cells among CD11c+MHCII− cells that contain preDCs (H) and surface CD135 expression among Xcr1Venus immature (MHCIIloCD24+XCR1+, left) or migratory (XCR1+CD8α−CD103+, right) cDC1s in LNs (I).Data display pooled data from ≥2 independent experiments (A–I) (A, n = 6–8; B and C, n = 3–4; D and E, n = 3; F, n = 4; G, n = 4–6; H and I, n = 4–6). Error bars indicate the mean ± SD. Comparison between groups was calculated using one-way ANOVA or unpaired Student’s t tests. ∗∗∗p value < 0.001, ∗∗p value < 0.01, ∗p value < 0.05.

Fig 4: Increased Flt3L signaling accelerates local cDC1 development(A and B) Prtn3LSL-TomXcr1WT or Prtn3LSL-TomXcr1DTR mice received DTx and tamoxifen according to the indicated scheme to analyze cDC1 development in LNs after cDC1 depletion. Frequency of MHCII− cells (A) and expression of MHCII and CD11c among Tomato+ immature/developing (MHCII−/loCD24+) cDC1s in LNs (B).(C) Prtn3LSL-TomXcr1WT or Prtn3LSL-TomXcr1DTR mice received DTx at −6 h, tamoxifen at 0 h, and analyzed 5 days after depletion, similar to the experimental setup in (A). Expression of MHCII and CD11c among Tomato+ immature (MHCIIloCD24+) cDC1s in LNs.(D) PreDCs from the BM of Zbtb46GFP mice were transferred into Xcr1WT or Xcr1DTR mice after depletion and analyzed 66 h after transfer. Expression of MHCII, CD11c and XCR1 among transferred GFP+ immature (MHCIIloCD24+XCR1+) cDC1s in LNs (right).(E and F) Analysis of LNs of Zbtb46GFP mice 16 h after i.p. Flt3L injection. Frequency of preDCs among immature/developing (MHCII−/lo) cDCs (left), cell numbers of preDCs (right) (E), and expression of MHCII, CD11c, and XCR1 among immature (MHCIIloCD24+XCR1+) cDC1s in LNs (F).(G and H) Analysis of draining LNs of Zbtb46GFP mice 16 h after s.c. Flt3L injection into the foot hock. Surface CD135 expression among preDCs (G) and surface CD135 and CD11c expression among immature (MHCIIloCD24+XCR1+) cDC1s in draining LNs (H).(I–K) Prtn3LSL-Tom mice received tamoxifen and Flt3L according to the indicated scheme. Frequency of MHCII− cells (I) and expression of MHCII and CD11c (J) and frequency of XCR1+ cells among Tomato+ immature/developing (MHCII−/loCD24+) cDC1s in LNs (K).Data display pooled data from ≥2 independent experiments (A–K) (A and B, n = 5–6; C, n = 13; D, n = 4–5; E and F, n = 3; G and H, n = 4; I–K, n = 7). Error bars indicate the mean ± SD. Comparison between groups was calculated using paired or unpaired Student’s t tests. ∗∗∗p value < 0.001, ∗∗p value < 0.01, ∗p value < 0.05.

Fig 5: SH deficiency enhances pDC-induced inhibition of CD4 T cells activation in vitro. Purified bone marrow FLT3L-derived pDCs from WT and SH KO mice (7.5 × 10e4 cells per well) were cocultured with purified naive CD4 T cells (0.3 million cells per well), followed by stimulation with CD3/CD28 microbeads for 72 hours. (A, B) Representative FACS plots of CD25+ and CD69+ CD4 T cells. Summarized data of the percentage of (C, D) CD25+ and CD69+ CD4 T cells, and (E, F) double CD69/CD25 positive or negative CD4 T cells. Enzyme-linked immunosorbent assay detection of (G, H) IFN-γ and IL21 concentrations in the culture supernatants. Bars in the scatter plots represent the median value. Statistical analysis was performed with 1-way ANOVA and Tukey post hoc test; ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005; ∗∗∗∗P < .00005.