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: 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).

Fig 4: 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+CD8a-CD103+, right) cDC1s (C) in draining LNs of Zbtb46GFP mice 24 h after s.c. IFNa injection into the foot hock.(D and E) Surface CD135 expression among preDCs (CD11c+MHCII-) (D) and immature (MHCIIloCD24+, left) or migratory (XCR1+CD8a-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. IFNa 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+CD8a-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 5: 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.