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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34 + SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9 + FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19 + and Cxcl9 + FRCs in LNs of Irf3 -/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs., (© 2022. The Author(s).)

Lymphatic transport of molecules and migration of myeloid cells to lymph nodes (LNs) continuously inform lymphocytes on changes in drained tissues. Here, using LN transplantation, single-cell RNA-seq, spectral flow cytometry, and a transgenic mouse model for photolabeling, we showed that tissue-derived unconventional T cells (UTCs) migrate via the lymphatic route to locally draining LNs. As each tissue harbored a distinct spectrum of UTCs with locally adapted differentiation states and distinct T cell receptor repertoires, every draining LN was thus populated by a distinctive tissue-determined mix of these lymphocytes. By making use of single UTC lineage-deficient mouse models, we found that UTCs functionally cooperated in interconnected units and generated and shaped characteristic innate and adaptive immune responses that differed between LNs that drained distinct tissues. Lymphatic migration of UTCs is, therefore, a key determinant of site-specific immunity initiated in distinct LNs with potential implications for vaccination strategies and immunotherapeutic approaches., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aims: Recent studies have revealed that B cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure-but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilization and activity in situ., Methods and Results: Histological, flow cytometry, and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B cells in the mediastinal lymph nodes. The repertoire of cardiac B cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed 'heart-associated B cells' (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5), and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on Days 1 and 5 post-MI. When compared with wild-type controls, mice treated with a neutralizing CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups., Conclusion: Our study reveals that polyclonal B cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3 + regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.