Fig 2: CXCL9/10+ DCs occupy distinct regions in the spleen(A) Spleens from REX3 mice were imaged 6 weeks after infection with T. gondii. Sections were stained with F4/80 to highlight the RP and B220 to highlight B cell follicles. The mean fluorescence intensity was measured for CXCL9-RFP and CXCL10-BFP in each splenic region. Data were normalized to the B cell zone to adjust for background fluorescence.(B and C) Spleens from WT mice infected for 6 weeks were stained with XCR1 (B) or 33D1 (C) together with CD11c to identify cDC-1s and cDC-2s, respectively. Summary data show mean fluorescence for the indicated marker in each splenic region. Data were normalized to the B cell follicle as in (A).(D and E) WT (D) and CXCR3 KO (E) TG6-GFP neonatal chimeras were stained with 33D1 to identify cDC-2 clusters. The density of GFP cells within cDC-2 clusters was then measured and normalized to the total density of GFP cells in the slice.Data in (A) are representative of three mice, and data in (B)–(E) are representative of two mice.

Fig 3: preDCs enter via medullary HEVs and develop in the LN medulla(A) Confocal microscopy showing Tomato+ monocytes, neutrophils, and cDCs in LNs of Prtn3LSL-TomZbtb46GFP mice 3 days after tamoxifen injection. Arrow shows a Tomato+GFP+ preDC in a medullary cord (left). Frequency of cells in medulla among monocytes, neutrophils, and cDCs (right).(B) Confocal microscopy showing GFP+ preDCs in LNs of WT mice 2 h after i.v. transfer of preDCs from the BM of Zbtb46GFP mice (left). Arrows show GFP+ preDCs in medullary cords (left). Frequency of cells in medulla among transferred preDCs and transferred T cells 2 h after i.v. transfer into WT (right).(C) Cell numbers of resident (CD8a+) or migratory (CD8a-CD103+) XCR1+ cDC1s in 6 LNs of Xcr1DTR mice at indicated time points after depletion of cDC1s with DTx injection.(D) Confocal microscopy showing XCR1+ cDC1s in LNs of non-depleted control and 3 days after depletion of cDC1s with DTx injection in Xcr1DTR/Venus mice. Representative images (left). Frequency of cells in paracortex among XCR1+ cDC1s (right).(E) Localization of XCR1+ cDC1s in medullary cords within the medulla in LNs of Xcr1Venus mice.(F) Analysis of LNs of Xcr1Venus mice 1 h after i.v. injection of anti-XCR1 antibody. Confocal microscopy showing i.v.-labeled cDC1s (left) and flow cytometric analysis showing percentages of i.v.-labeled cells among resident (CD8a+) or migratory (CD8a-CD103+) XCR1+ cDC1s (right).(G) Ex vivo photoconversion of the paracortex of LN slices from Dendra2 mice and the subsequent analysis of these photoconverted LN slices via flow cytometry. Representative image after photoconversion (left top), flow cytometric analysis showing frequency of D-Red+ converted cells among XCR1+ cDC1s (left bottom) and percentage of CD8a+ cells among D-Red+ or D-Red- XCR1+ cDC1s (right).(H) Confocal microscopy showing CD8a+ XCR1+ cDC1s in LN of Xcr1Venus mice after depletion of CD8ß+ cells with antibody injection.Images are representative of =2 independent experiments (A, B, and D–H) and data display pooled data from =2 independent experiments (A–D, F, and G) (A, n = 5 using 2 LNs/n; B, n = 3–4 using 1–6 LNs/n; C, n = 4–6; D, n = 4–8 using 1–4 LNs/n; E, n = 3; F, n = 3 using 1–2 LNs/n; G, n = 4 using 2–4 LNs/n; H, n = 3 using 1–3 LNs/n). Error bars indicate the mean ± SD. Comparison between groups was calculated using one-way ANOVA, paired or unpaired Student’s t tests. ***p value < 0.001, **p value < 0.01, *p value < 0.05. Scale bars: 20 µm in (A) and (B); 30 µm in (E); and 200 µm in (D) and (F)–(H).

Fig 4: Synchronized migration of resident cDC1s during inflammation impairs LN functionality(A) Flow cytometric analysis draining popliteal LNs of WT mice 16, 24, and 40 h after s.c. infection with MVA into the foot hock. Frequency of CD86+CCR7+ cells among resident (XCR1+CD8a+) cDC1s (left) and mean fluorescence intensity (MFI) values of CD86 and CCR7 among CD86+CCR7+ resident cDC1s (right).(B) Localization of XCR1+ cDC1s in popliteal LNs of Xcr1Venus mice 24 h after s.c. infection with MVA or IFNa injection into the foot hock. Confocal microscopy (left), density plots showing the distribution of XCR1+ cDC1s (middle, outer black line outlines the LN border and inner black line outlines the paracortex), and frequency of XCR1+ cDC1s in the medulla (right).(C–G) WT mice were injected with IFNa or PBS s.c. into the foot hock and 24 h later AF647-labeled BSA was injected s.c. into the foot hock and draining LNs were analyzed 2 h after BSA injection. Experimental setup (C), frequency of BSA+ cells among resident (XCR1+CD8a+) cDC1s (left), and number of BSA+ resident cDC1s (right) (D), BSA intensity among BSA+ resident cDC1s (E), frequency of BSA+ cells among immature (MHCIIloCD24+) cDC1s (left) and number of BSA+ immature cDC1s (right) (F), and BSA intensity among BSA+ immature cDC1s (G).Images are representative of =2 independent experiments (B) and data display pooled data from =2 independent experiments (A–G) (A, n = 6–8; B, n = 4–6 using 2 LNs/n; C–G, n = 3). 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. Scale bars represent 200 µm in (B).

Fig 5: Resident cDC1s migrate from medulla toward paracortex during their development(A) Frequency of Tomato+ cells among MHCIIlo or MHCIIhi XCR1+ cDC1s from LNs of Mki67LSL-Tom mice treated with tamoxifen 40 h before sacrifice.(B) Localization of XCR1+ cDC1s in LNs of Mki67LSL-TomXcr1Venus mice treated with tamoxifen 40 h before sacrifice. Confocal microscopy (left top), localization of the indicated XCR1+ cDC1 populations in a representative LN (left bottom, outer gray line outlines the LN border and inner gray line outlines the paracortex) and frequency of cells in the paracortex among the indicated populations.(C) Frequency of cells in the paracortex among Ki67+ or Ki67- XCR1+ cDC1s in LNs of Xcr1DTR/Venus mice 3 days after depletion of cDC1s with DTx injection.(D) Intravital microscopy images showing local proliferation of XCR1+ cDC1s in the LN medulla of Xcr1DTR/Venus mice 68 h after depletion of cDC1s with DTx. White arrows show proliferating cDC1s in medulla.(E) Confocal microscopy of vibratome slices XCR1+ cDC1s around blood vessels in LNs of Xcr1DTR/Venus mice 3 days after depletion. Yellow lines outline a vessel extending from medulla to paracortex and images show the same spot at different depth/z stack.(F and G) Mixed WT:Ccr7-/- (85:15) BM chimeras were injected i.v. aXCR1 antibody 1 h before sacrifice (n = 5 mice in 2 experiments). Experimental setup (left) and frequency of ivXCR1+ cells among WT or Ccr7-/- (KO) cells for MHCIIlo and MHCIIhi cells in resident (XCR1+CD8a+) cDC1s (right) (F). Frequency of MHCIIhi cells among WT or Ccr7-/- (KO) resident (XCR1+CD8a+) cDC1s (G).Images are representative of =2 independent experiments (B, D, and E) and data display pooled data from =2 independent experiments (A–C, F, and G) (A, n = 4; B, n = 3 using 3–4 LNs/n; C, n = 3 using 3–4 LNs/n; D, n = 4 using 1–2 LNs/n; E, n = 3 using 1–3 LNs/n; F and G, n = 5). Error bars indicate the mean ± SD. Comparison between groups was calculated using one-way ANOVA or paired Student’s t tests. ***p value < 0.001, *p value < 0.05. Scale bars: 20 µm in (B); 30 µm in (D); and 100 µm in (E).