Chemokines and adhesion molecules upregulated in lymphatic endothelial cells (LECs) during tissue inflammation are thought to enhance dendritic cell (DC) migration to draining lymph nodes, but the in vivo control of this process is not well understood. We performed a transcriptional profiling analysis of LECs isolated from murine skin and found that inflammation induced by a contact hypersensitivity (

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3−/− mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing redfluorescent vasculature and yellowfluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steadystate and inflammation. (Blood. 2012; 120(11):2249-2258) IntroductionDendritic cells (DCs) are important in the initiation of adaptive immune responses. In peripheral tissues, such as the skin, DCs take up antigen, mature, and migrate via lymphatic vessels (LVs) to draining lymph nodes (dLNs), where they present antigen to resting T cells. Although this migration pattern has been known for more than 20 years, 1 DC migration into LVs is only now starting to be unraveled at the cellular level using live imaging technologies. [2][3][4] Before transmigrating across the lymphatic endothelium, DCs first need to squeeze through preformed, narrow pores present in the lymphatic basement membrane (BM). 3 DC entry into LVs is thought to occur at the level of primary lymphatic capillaries, which feature discontinuous cell junctions with "button"-like accumulations of cell adhesion molecules. 5 At the sites of such buttons, lymphatic endothelial cells (LECs) partially overlap and generate open flaps, through which leukocytes enter into lymphatics. 3,5 The most prominent mediator of DC migration into afferent lymphatics is CCL21, a chemokine constitutively expressed in LVs. 6,7 More recently, also other LEC-expressed molecules that mediate DC migration via LVs to dLNs have been identified. [8][9][10][11] Notably, ICAM-1 and VCAM-1, which are up-regulated in LVs during inflammation, 8,12 have been implicated in this process. 8 Intriguingly, experiments performed with pan-integrin knockout DCs revealed that DC migration to dLNs in steady-state was integrin-independent. 2 This ...

Systemic high-dose IL2 promotes long-term survival in a subset of metastatic melanoma patients, but this treatment is accompanied by severe toxicities. The immunocytokine L19-IL2, in which IL2 is fused to the human L19 antibody capable of selective accumulation on tumor neovasculature, has recently shown encouraging clinical activity in patients with metastatic melanoma. In this study, we have investigated the therapeutic performance of L19-IL2, administered systemically in combination with a murine anti-CTLA-4 antibody or with a second clinical-stage immunocytokine (L19-TNF) in two syngeneic immunocompetent mouse models of cancer. We observed complete tumor eradications when L19-IL2 was used in combination with CTLA-4 blockade. Interestingly, mice cured from F9 tumors developed new lesions when rechallenged with tumor cells after therapy, whereas mice cured from CT26 tumors were resistant to tumor rechallenge. Similarly, L19-IL2 induced complete remissions when administered in a single intratumoral injection in combination with L19-TNF, whereas the two components did not lead to cures when administered as single agents. These findings provide a rationale for combination trials in melanoma, as the individual therapeutic agents have been extensively studied in clinical trials, and the antigen recognized by the L19 antibody has an identical sequence in mouse and man.