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.

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 burgeoning field of leukocyte trafficking has created new and exciting opportunities in the clinic. Trafficking signals are being defined that finely control the movement of distinct subsets of immune cells into and out of specific tissues. Because the accumulation of leukocytes in tissues contributes to a wide variety of diseases, these 'molecular codes' have provided new targets for inhibiting tissue-specific inflammation, which have been confirmed in the clinic. However, immune cell migration is also critically important for the delivery of protective immune responses to tissues. Thus, the challenge for the future will be to identify the trafficking molecules that will most specifically inhibit the key subsets of cells that drive disease processes without affecting the migration and function of leukocytes required for protective immunity.

Over the past decades, intravital microscopy (IVM), the imaging of cells in living organisms, has become a valuable tool for studying the molecular determinants of lymphocyte trafficking. Recent advances in microscopy now make it possible to image cell migration and cell-cell interactions in vivo deep within intact tissues. Here, we summarize the principal techniques that are currently used in IVM, discuss options and tools for fluorescence-based visualization of lymphocytes in microvessels and tissues, and describe IVM models used to explore lymphoid and non-lymphoid organs. The latter will be introduced according to the physiologic itinerary of developing and differentiating T and B lymphocytes as they traffic through the body, beginning with their development in bone marrow and thymus and continuing with their migration to secondary lymphoid organs and peripheral tissues.

It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function-associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1. These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that primes LFA-1 for ligand binding and firm adhesion.

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules leads to rolling and is critical for recruitment of lymphocytes into peripheral lymph nodes. Peripheral node addressin represents a class of L-selectin ligands recognized by the function-blocking monoclonal antibody MECA-79. Its epitope overlaps with sialyl 6-sulfo Lewis X, an L-selectin recognition determinant. Here, mice lacking two N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6ST-1 and GlcNAc6ST-2) demonstrated elimination of both peripheral node addressin and sialyl 6-sulfo Lewis X in high endothelial venules, considerably reduced lymphocyte homing to peripheral lymph nodes and reduced sticking of lymphocytes along high endothelial venules. Our results establish an essential function for the sulfotransferases in L-selectin ligand synthesis and may have relevance for therapy of inflammatory diseases.

Mature recirculating B cells are generally assumed to exist in follicular niches in secondary lymphoid organs, and these cells mediate T-dependent humoral immune responses. We show here that a large proportion of mature B lymphocytes occupy an anatomically and functionally distinct perisinusoidal niche in the bone marrow. Perisinusoidal B cells circulate freely, as revealed by parabiosis studies. However, unlike their counterparts in the follicular niche, these cells are capable of being activated in situ by blood-borne microbes in a T-independent manner to generate specific IgM antibodies. The bone marrow represents a unique type of secondary lymphoid organ in which mature B cells are strategically positioned in the path of circulating microbes.

T cell activation by intestinal dendritic cells (DC) induces gut-tropism. We show that, reciprocally, DC from peripheral lymph nodes (PLN-DC) induce homing receptors promoting CD8 T cell accumulation in inflamed skin, particularly ligands for P- and E-selectin. Differential imprinting of tissue-tropism was independent of Th1/Th2 cytokines and not restricted to particular DC subsets. Fixed PLN-DC retained the capacity to induce selectin ligands on T cells, which was suppressed by addition of live intestinal DC. By contrast, fixed intestinal DC failed to promote gut-tropism and instead induced skin-homing receptors. Moreover, the induction of selectin ligands driven by antigen-pulsed PLN-DC could be suppressed "in trans" by adding live intestinal DC, but PLN-DC did not suppress gut-homing receptors induced by intestinal DC. Reactivation of tissue-committed memory cells modified their tissue-tropism according to the last activating DC's origin. Thus, CD8 T cells activated by DC acquire selectin ligands by default unless they encounter fixation-sensitive signal(s) for gut-tropism from intestinal DC. Memory T cells remain responsive to these signals, allowing for dynamic migratory reprogramming by skin- and gut-associated DC.

Sphingosine-1-phosphate (S1P) and its receptor S1P1 control T-cell egress from thymus and secondary lymphoid organs (SLOs). To further define the role of S1P1 in lymphocyte trafficking, we performed adoptive transfer experiments and intravital microscopy (IVM) using both S1P1-/- lymphocytes and recipient wild-type (WT) mice treated with FTY720, an immunosuppressant that downmodulates S1P receptors. S1P1 deficiency and FTY720 caused rapid disappearance of T cells from blood, prolonged retention in SLOs, and accumulation in bone marrow, but did not alter interstitial T-cell motility in peripheral lymph nodes (PLNs) as assessed by multiphoton IVM. However, S1P1-/- lymphocytes displayed reduced short-term homing to PLNs due to attenuated integrin-mediated firm arrest in high endothelial venules (HEVs). By contrast, S1P1-/- T cells homed normally to Peyer patches (PPs), whereas S1P1-/- B cells had a marked defect in homing to PPs and arrested poorly in PP HEVs. Therefore, S1P1 not only controls lymphocyte egress from SLOs, but also facilitates in a tissue- and subset-specific fashion integrin activation during homing. Interestingly, FTY720 treatment enhanced accumulation of both S1P1 sufficient and S1P1-/- T cells in PPs by enhancing integrin-mediated arrest in HEVs. Thus, FTY720 exerts unique effects on T-cell traffic in PPs that are independent of T-cell-expressed S1P1.

Intact cellular migration is critically important for the induction and regulation of the immune response. The Wiskott-Aldrich syndrome protein (WASP) regulates surface receptor signaling to the actin cytoskeleton in hematopoietic cells and thus plays a pivotal role in cellular locomotion. WASP deficiency causes the Wiskott-Aldrich syndrome (WAS), characterized by immunodeficiency, thrombocytopenia, and eczema. Cell migration defects may contribute to the pathophysiology of WAS. In this study, we used a variety of in vitro and in vivo assays to comprehensively analyze migration properties of lymphocytes, dendritic cells (DC), and neutrophils from WASP-deficient mice. We provide evidence that WASP-deficient lymphocytes show a marked reduction in tethering in an in vitro flow chamber assay as well as decreased migration of T cells in response to the CC chemokine ligand 19 (CCL19). In vivo, compared with wild-type lymphocytes, WASP-deficient lymphocytes showed significantly impaired homing to Peyer's patches upon adoptive transfer into recipient mice. In addition, bone marrow-derived DC migrated less efficiently in response to CCL19. In vivo studies showed decreased migration of DC from skin to draining lymph nodes in WASP-deficient animals. Finally, we also document decreased neutrophil migration in vitro and in vivo. In summary, our studies suggest that WASP plays an important role in the locomotion of lymphocytes, DC, and granulocytes in vitro and in vivo and thus, reveal a crucial role of WASP in physiological trafficking of various hematopoietic cell lineages. These results further delineate immunological abnormalities in WASP-deficient mice, which will be useful to assess preclinical gene therapy studies.