Whereas naive T cells migrate only to secondary lymphoid organs, activation by antigen confers to T cells the ability to home to non-lymphoid sites. Activated effector/memory T cells migrate preferentially to tissues that are connected to the secondary lymphoid organs where antigen was first encountered. Thus, oral antigens induce effector/memory cells that express essential receptors for intestinal homing, namely the integrin alpha4beta7 and CCR9, the receptor for the gut-associated chemokine TECK/CCL25 (refs 6, 8, 9). Here we show that this imprinting of gut tropism is mediated by dendritic cells from Peyer's patches. Stimulation of CD8-expressing T cells by dendritic cells from Peyer's patches, peripheral lymph nodes and spleen induced equivalent activation markers and effector activity in T cells, but only Peyer's patch dendritic cells induced high levels of alpha4beta7, responsiveness to TECK and the ability to home to the small intestine. These findings establish that Peyer's patch dendritic cells imprint gut-homing specificity on T cells, and thus license effector/memory cells to access anatomical sites most likely to contain their cognate antigen.

Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.

The dermis contains a novel population of γδT cells that are distinct from epidermal γδT cells and produce IL-17 in response to mycobacterial infection.

The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (≥30 min), cognate antigen–dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.

Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Gαi protein-coupled receptor–dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.

Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space.

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.