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Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses 1 , 2 . Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood 3 , 4 . Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies. Scheiermann and colleagues show that circadian clocks control the infiltration of dendritic cells into skin lymphatics in mice and humans, with a peak migration to the lymph nodes during the rest phase.

In rheumatoid arthritis (RA), synovial tissue abundantly expresses CCL21, a chemokine strongly associated with RA susceptibility. In this study, we aimed to characterize the functional significance of CCL21/CCR7 signaling in different phases of RA pathogenesis. We determined that CCR7 is a hallmark of RA M1 synovial fluid (SF) macrophages, and its expression in RA monocytes and in vitro differentiated macrophages is closely associated with disease activity score (DAS28). In early stages of RA, monocytes infiltrate the synovial tissue. However, blockade of SF CCL21 or CCR7 prevents RA SF-mediated monocyte migration. CCR7 expression in the newly migrated macrophages can be accentuated by LPS and IFNγ and suppressed by IL-4 treatment. We also uncovered that CCL21 stimulation increases the number of M1-polarized macrophages (CD14+CD86+), resulting in elevated transcription of IL-6 and IL-23. These CCL21-induced M1 cytokines differentiate naïve T cells to Th17 cells, without affecting Th1 cell polarization. In the erosive stages of disease, CCL21 potentiates RA osteoclastogenesis through M1-driven Th17 polarization. Disruption of this intricate crosstalk, by blocking IL-6, IL-23, or IL-17 function, impairs the osteoclastogenic capacity of CCL21. Consistent with our in vitro findings, we establish that arthritis mediated by CCL21 expands the joint inflammation to bone erosion by connecting the differentiation of M1 macrophages with Th17 cells. Disease progression is further exacerbated by CCL21-induced neovascularization. We conclude that CCL21 is an attractive novel target for RA therapy, as blockade of its function may abrogate erosive arthritis modulated by M1 macrophages and Th17 cell crosstalk.

Hashimoto’s thyroiditis (HT) is the most common autoimmune disease characterized by lymphocytic infiltration and thyrocyte destruction. Dissection of the interaction between the thyroidal stromal microenvironment and the infiltrating immune cells might lead to a better understanding of HT pathogenesis. Here we show, using single-cell RNA-sequencing, that three thyroidal stromal cell subsets, ACKR1 + endothelial cells and CCL21 + myofibroblasts and CCL21 + fibroblasts, contribute to the thyroidal tissue microenvironment in HT. These cell types occupy distinct histological locations within the thyroid gland. Our experiments suggest that they might facilitate lymphocyte trafficking from the blood to thyroid tissues, and T cell zone CCL21 + fibroblasts may also promote the formation of tertiary lymphoid organs characteristic to HT. Our study also demonstrates the presence of inflammatory macrophages and dendritic cells expressing high levels of IL-1β in the thyroid, which may contribute to thyrocyte destruction in HT patients. Our findings thus provide a deeper insight into the cellular interactions that might prompt the pathogenesis of HT. Hashimoto’s Thyroiditis is an autoimmune disease with a complex pathomechanism. Authors here show by single cell RNA sequencing that the thyroidal microenvironment in the disease is characterised by three stromal cell subtypes that are potentially responsible for the recruitment of infiltrating inflammatory immune cells, such as macrophages and dendritic cells.

Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial–mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-β1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-β1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-β1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-β1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells.

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor‐mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7‐dependent signaling in MDA‐MB‐231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7‐dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA‐MB‐231–derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE‐1+ intratumoral and peritumoral lymphatic vessels. In the three‐dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21‐reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand–expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis. Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. We here demonstrated that CXCL12/CXCR4 signaling enhances breast cancer cell response at lower concentrations of CCR7 ligand chemokine. In the tumor microenvironment, CXCL12 may indirectly regulate lymph node metastasis by promoting CCR7 signaling in cancer cells.

The presence of lymph node (LN)-like vasculature in tumours, characterized by expression of peripheral node addressin and chemokine CCL21, is correlated with T-cell infiltration and positive prognosis in breast cancer and melanoma patients. However, mechanisms controlling the development of LN-like vasculature and how it might contribute to a beneficial outcome for cancer patients are unknown. Here we demonstrate that LN-like vasculature is present in murine models of melanoma and lung carcinoma. It enables infiltration by naive T cells that significantly delay tumour outgrowth after intratumoral activation. Development of this vasculature is controlled by a mechanism involving effector CD8 T cells and NK cells that secrete LTα 3 and IFNγ. LN-like vasculature is also associated with organized aggregates of B lymphocytes and gp38 + fibroblasts, which resemble tertiary lymphoid organs that develop in models of chronic inflammation. These results establish LN-like vasculature as both a consequence of and key contributor to anti-tumour immunity. The presence of lymph node-like vasculature in tumours correlates with positive prognosis, but the mechanisms by which it forms and affects tumour growth are unclear. Here the authors show that it is induced by CD8 and NK cells, and supports naive T cells’ differentiation into antitumour effectors.

Dendritic cell (DC) homing to the lymphatics and positioning within the lymph node is important for adaptive immunity, and is regulated by gradients of CCL19 and CCL21, ligands for CCR7. Despite the importance of DC chemotaxis, it is not well understood how DCs interpret gradients of these chemokines in a complex 3D microenvironment. Here, we use a microfluidic device that allows rapid establishment of stable gradients in 3D matrices to show that DC chemotaxis in 3D can respond to CCR7 ligand gradients as small as 0.4%, which helps explain how DCs sense lymphatic vessels in an environment where broadcast distance for chemokine diffusion is hindered by convective flows into the vessel. Interestingly, DCs displayed similar sensitivities to both chemokines at small gradients ([less-than or equal to] 60 nM/mm), but migrated more efficiently towards higher gradients of CCL21, which unlike CCL19 binds strongly to matrix proteoglycans and signals without the need for internalization. Furthermore, cells preferentially migrated towards CCL21 when exposed to equal and opposite gradients of CCL21 and CCL19 simultaneously, even when matrix-binding of CCL21 was prevented. Although these ligands have similar binding affinity to CCR7, our results demonstrate that, in a 3D environment, CCL21 is a more potent directional cue for DC migration than CCL19. These findings provide new quantitative insight into DC chemotaxis in a physiological 3D environment and suggest how CCL19 and CCL21 may signal differently to fine-tune DC homing and positioning within the lymphatic system. These results also have broad relevance to other systems of cell chemotaxis, which remain poorly understood in the 3D context.

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130–4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623–630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341–3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

Migration and homing of immune cells are critical for immune surveillance. Trafficking is mediated by combinations of adhesion and chemokine receptors that guide immune cells, in response to chemokine signals, to specific locations within tissues and the lymphatic system to support tissue-localized immune reactions and systemic immunity 1 , 2 . Here we show that disruption of leukaemia inhibitory factor (LIF) production from group 2 innate lymphoid cells (ILC2s) prevents immune cells leaving the lungs to migrate to the lymph nodes (LNs). In the absence of LIF, viral infection leads to plasmacytoid dendritic cells (pDCs) becoming retained in the lungs where they improve tissue-localized, antiviral immunity, whereas chronic pulmonary allergen challenge leads to marked immune cell accumulation and the formation of tertiary lymphoid structures in the lung. In both cases immune cells fail to migrate to the lymphatics, leading to highly compromised LN reactions. Mechanistically, ILC2-derived LIF induces the production of the chemokine CCL21 from lymphatic endothelial cells lining the pulmonary lymphatic vessels, thus licensing the homing of CCR7 + immune cells (including dendritic cells) to LNs. Consequently, ILC2-derived LIF dictates the egress of immune cells from the lungs to regulate tissue-localized versus systemic immunity and the balance between allergen and viral responsiveness in the lungs. Disruption of leukaemia inhibitory factor production from group 2 innate lymphoid cells prevents immune cells leaving the lungs to migrate to lymph nodes, leading to plasmacytoid dendritic cells becoming retained in the lungs following viral infection.

Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoid-like reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and even nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, which facilitates tumor progression.