Fig 2: 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 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: Hyperactivating Stimuli Eradicate Established Tumors That Are Resistant to Checkpoint InhibitorsMice of the indicated genotypes were inoculated subcutaneously on the left upper back with (A) 5 × 105 of MC38OVA cells, (B) 3 × 105 B16OVA cells, (C) 3 × 105 B16-F10 cells, (D) 3 × 105 CT26 cells, or (E and F) 3 × 105 LLC1 cells. In (A)–(E), when tumors reached 3–4 mm in size, mice were either left untreated (unimmunized) or were injected s.c. on the right flank with WTLs plus LPS and PGPC with or without neutralizing anti-IL-1ß antibodies, anti-CD4, anti-CD8a antibodies, or (F) IL-RA. Mice received two boost injections with WTLs and LPS plus PGPC. In (B)–(E), alternatively, tumor-bearing mice were injected with anti-PD-1 antibody. The percentage of survival is indicated (n = 10–12 mice per group). In (F), LLC1 tumors were harvested at the endpoint of tumor growth. The percentage of CD8+ TILs (left panel) among CD3+CD45+ live cells and CD69+CD103+ TRM cells among CD8+ TILs (middle panel) were measured by flow cytometry. CD45+ live TILs were cultured for 48 h on anti-CD28 and anti-CD3 coated plates. IFN? was measured by ELISA (right panel) (n = 5 mice per group). p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or = 0.0001 (****) are indicated.