Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks

Abstract

Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.

Keywords: CD135; Flt3L; Prtn3; cDC1; conveyor belt; feedback; infection; inflammation; migration; preDC.

Graphical abstract

Figure 1

Fully functional resident cDC1s gradually develop from preDCs over several days (A) Representative gating strategies for cDC1s in LNs of WT mice. MHCII^loCD24⁺ gate shows immature cDC1s (top) and XCR1⁺CD8α⁺ gate shows resident cDC1s (immature and mature) (bottom). (B and C) Analysis of immature cDC1 (MHCII^loCD24⁺) repopulation in LNs of Xcr1^DTR mice 2, 3, or 4 days after cDC1 depletion. Expression levels of XCR1-Venus reporter, CD8α, CD205, and CD81 (B) and multidimensional tSNE analysis of flow cytometric data using 13 markers (C). (D and E) Analysis of newly developing unconverted (K-Red⁻K-Green⁺) immature cDC1s (MHCII^loCD24⁺XCR1⁺) after transdermal photoconversion of inguinal LNs in Xcr1^KikGR mice. Frequency of unconverted (K-Red⁻K-Green⁺) cells among immature cDC1s (MHCII^loCD24⁺XCR1⁺) (D) and expression levels of XCR1, CD8α, and CD226 among unconverted (K-Red⁻K-Green⁺) immature cDC1s (MHCII^loCD24⁺XCR1⁺) (E). (F) Analysis of antigen uptake by immature cDC1s (MHCII^loCD24⁺) in draining LNs of WT mice 2 h after s.c. injection of BSA-AF647 (bovine serum albumin) into the foot hock. Frequency of BSA-AF647⁺ cells among XCR1⁻ vs. XCR1⁺ (left) and CD81⁻ vs. CD81⁺ cells (right). Data display one representative of ≥2 independent experiments (A and C) or pooled data from ≥2 independent experiments (B and D–F) (A, n ≥ 3; B and C, n = 4–6; D and E, n = 4–7 using 8–14 LNs; F, n = 5). 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.

Figure 2

Prtn3-based fate-tracking reveals early phases of DC development in LNs (A) Expression of the human CD4 reporter among the indicated populations in the BM and LNs of Prtn3^CreERT2-hCD4 mice. Black lines show WT controls. CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; GP, granulocyte progenitor; MDP, monocyte-dendritic cell progenitor; cMoP, common monocyte progenitor; Mono, monocytes; Neutro, neutrophils; pDC, plasmacytoid dendritic cell. (B) Diagram showing the labeling/fate-tracking of a wave of myeloid precursors in the BM of Prtn3^LSL-Tom mice after tamoxifen injection and the consequent migration to LNs. (C) Frequency of Tomato⁺ cells among the indicated populations in the BM and LNs of Prtn3^LSL-Tom mice 24 h after tamoxifen injection. (D) Frequency of Tomato⁺ cells among the indicated populations in the BM of Prtn3^LSL-Tom mice 1, 3, 5, and 7 days after tamoxifen injection. (E) Frequency of MHCII⁻, MHCII^lo, and MHCII^hi cells among all (top) or Tomato⁺ (bottom) cDCs (Lin⁻(Ly6C^hiCD11b⁺)^–B220⁻CD11c⁺) in LNs of Prtn3^LSL-Tom mice 3, 5, and 7 days after tamoxifen injection. (F–L) Single-cell RNA sequencing (scRNA-seq) analysis of preDCs in LNs. (F) Experimental setup. PreDCs (CD11c⁺MHCII⁻CD135⁺) from LNs of unmanipulated WT mice and Tomato⁺ preDCs from LNs of Prtn3^LSL-Tom mice that received tamoxifen injection 3, 5, or 7 days earlier were sorted and analyzed by scRNA-seq. (G) Uniform manifold approximation and projection (UMAP) plot displaying 8,516 scRNA-seq transcriptomes clustered in 10 different clusters. Lines indicate different lineage trajectories calculated by the Slingshot algorithm starting from cluster 5. (H) UMAP plots displaying expression of Cd24a, Cx3cr1, Sirpa, and Ly6d genes. (I) Dotplot of representative marker genes associated with the identified clusters. Color indicates the Z score mean of the expression values across clusters and dot size represents fraction of cells in the cluster expressing the respective genes. (J) UMAP plot showing the distribution of Tomato⁺ preDCs among the clusters 3, 5, or 7 days after tamoxifen injection. (K) Normalized percentages of Tomato⁺ cells within the cDC1 lineage associated clusters (5, 2, and 9) 3, 5, or 7 days after tamoxifen injections. (L) Gene ontology terms enriched among genes that are upregulated during the early development of the cDC1 lineage in LNs. FDR, false discovery rate. Data display one representative of ≥2 independent experiments (A–D), pooled data from ≥2 independent experiments (E) or data from 1 experiment (F–L) (A, C, and D, n = 4; E, n = 6). Error bars indicate the mean ± SEM.

Figure 3

preDCs enter via medullary HEVs and develop in the LN medulla (A) Confocal microscopy showing Tomato⁺ monocytes, neutrophils, and cDCs in LNs of Prtn3^LSL-TomZbtb46^GFP 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 Zbtb46^GFP 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 (CD8α⁺) or migratory (CD8α⁻CD103⁺) XCR1⁺ cDC1s in 6 LNs of Xcr1^DTR 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 Xcr1^DTR/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 Xcr1^Venus mice. (F) Analysis of LNs of Xcr1^Venus 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 (CD8α⁺) or migratory (CD8α⁻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 CD8α⁺ cells among D-Red⁺ or D-Red⁻ XCR1⁺ cDC1s (right). (H) Confocal microscopy showing CD8α⁺ XCR1⁺ cDC1s in LN of Xcr1^Venus mice after depletion of CD8β⁺ 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).

Figure 4

Resident cDC1s migrate from medulla toward paracortex during their development (A) Frequency of Tomato⁺ cells among MHCII^lo or MHCII^hi XCR1⁺ cDC1s from LNs of Mki67^LSL-Tom mice treated with tamoxifen 40 h before sacrifice. (B) Localization of XCR1⁺ cDC1s in LNs of Mki67^LSL-TomXcr1^Venus 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 Xcr1^DTR/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 Xcr1^DTR/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 Xcr1^DTR/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. αXCR1 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 MHCII^lo and MHCII^hi cells in resident (XCR1⁺CD8α⁺) cDC1s (right) (F). Frequency of MHCII^hi cells among WT or Ccr7^−/− (KO) resident (XCR1⁺CD8α⁺) 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).

Figure 5

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⁺CD8α⁺) 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 Xcr1^Venus mice 24 h after s.c. infection with MVA or IFNα 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 IFNα 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⁺CD8α⁺) cDC1s (left), and number of BSA⁺ resident cDC1s (right) (D), BSA intensity among BSA⁺ resident cDC1s (E), frequency of BSA⁺ cells among immature (MHCII^loCD24⁺) 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).

Figure 6

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 (MHCII^loCD24⁺, left) or migratory (XCR1⁺CD8α⁻CD103⁺, right) cDC1s (C) in draining LNs of Zbtb46^GFP 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 (MHCII^loCD24⁺, left) or migratory (XCR1⁺CD8α⁻CD103⁺, right) cDC1s (E) in draining LNs of Zbtb46^GFP mice 16 h after i.p. Flt3L injection. (F) Surface CD135 expression among preDCs (CD11c⁺MHCII⁻) (left) and immature (MHCII^loCD24⁺, 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 Xcr1^DTR mice 2, 3, and 4 days after cDC1 depletion with DTx injection. (H and I) Mixed Xcr1^DTR:Xcr1^Venus (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 Xcr1^Venus immature (MHCII^loCD24⁺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.

Figure 7

Increased Flt3L signaling accelerates local cDC1 development (A and B) Prtn3^LSL-TomXcr1^WT or Prtn3^LSL-TomXcr1^DTR 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) Prtn3^LSL-TomXcr1^WT or Prtn3^LSL-TomXcr1^DTR 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 (MHCII^loCD24⁺) cDC1s in LNs. (D) PreDCs from the BM of Zbtb46^GFP mice were transferred into Xcr1^WT or Xcr1^DTR mice after depletion and analyzed 66 h after transfer. Expression of MHCII, CD11c and XCR1 among transferred GFP⁺ immature (MHCII^loCD24⁺XCR1⁺) cDC1s in LNs (right). (E and F) Analysis of LNs of Zbtb46^GFP 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 (MHCII^loCD24⁺XCR1⁺) cDC1s in LNs (F). (G and H) Analysis of draining LNs of Zbtb46^GFP 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 (MHCII^loCD24⁺XCR1⁺) cDC1s in draining LNs (H). (I–K) Prtn3^LSL-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.