The mechanism of B cell-antigen encounter in lymphoid tissues is incompletely understood. It is also unclear how immune complexes are transported to follicular dendritic cells. Here, using real-time two-photon microscopy we noted rapid delivery of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells captured immune complexes by a complement receptor-dependent mechanism from macrophage processes that penetrated the follicle and transported the complexes to follicular dendritic cells. Furthermore, cognate B cells captured antigen-containing immune complexes from macrophage processes and migrated to the T zone. Our findings identify macrophages lining the subcapsular sinus as an important site of B cell encounter with immune complexes and show that intrafollicular B cell migration facilitates the transport of immune complexes as well as encounters with cognate antigen.

Lymphocyte motility in lymph nodes is regulated by chemokines, but the contribution of integrins to this motility remains obscure. Here we examined lymphocyte migration over CCR7-binding chemokines that 'decorate' lymph node stroma. In a shear-free environment, surface-bound lymph node chemokines but not their soluble counterparts promoted robust and sustained T lymphocyte motility. The chemokine CCL21 induced compartmentalized clustering of the integrins LFA-1 and VLA-4 in motile lymphocytes, but both integrins remained nonadhesive to ligands on lymphocytes, dendritic cells and stroma. The application of shear stress to lymphocytes interacting with CCL21 and integrin ligands promoted robust integrin-mediated adhesion. Thus, lymph node chemokines that promote motility and strongly activate lymphocyte integrins under shear forces fail to stimulate stable integrin adhesiveness in extravascular shear-free environments.

Lymphocyte egress from lymph nodes (LNs) is dependent on sphingosine-1-phosphate (S1P), but the cellular source of this S1P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-1 CRE-mediated ablation of sphingosine kinase (Sphk) 1 and lacking Sphk2 have a loss of S1P in lymph while maintaining normal plasma S1P. In Lyve-1 Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Gαi-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell–cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S1P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S1P in lymphatic vessel maturation.

Mice carrying the recessive peripheral T cell deficiency (Ptcd) locus have a block in thymic egress but the mechanism responsible is undefined. Here we found that Ptcd T cells have an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus revealed an E26K point mutation within the actin regulator coronin-1A (Coro1a) that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. Discovery of another Coro1a mutant during an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for T cell lymphopenic mice prompted us to evaluate a T cell-deficient, B cell- and NK cell-sufficient (T−B+NK+) severe combined immunodeficiency (SCID) patient, whom we found had mutations in both CORO1A alleles. These findings establish a role for coronin-1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation, and reveal actin regulation as a biological process defective in human and mouse SCID.

The prominent display of opsonized antigen by follicular dendritic cells (FDCs) has long favored the view that they serve as antigen-presenting cells for B cells. Surprisingly, however, although B cell capture of antigen from macrophages and dendritic cells has been visualized, acquisition from FDCs has not been directly observed. Using two-photon microscopy, we visualized B cell capture of cognate antigen from FDCs. B cell CXCR5 expression was required, and encounter with FDC-associated antigen could be detected for >1 wk after immunization. B cell–FDC contact times were often brief but occasionally persisted for >30 min, and B cells sometimes acquired antigen together with FDC surface proteins. These observations establish that FDCs can serve as sites of B cell antigen capture, with their prolonged display time ensuring that even rare B cells have the chance of antigen encounter, and they suggest possible information transfer from antigen-presenting cell to B cell.

The splenic marginal zone (MZ) is a unique microenvironment where resident immune cells are exposed to the open blood circulation1,2. Despite its importance in responses against blood-borne antigens, lymphocyte migration in the MZ has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a 2-photon microscopy procedure to study MZ and follicular (FO) B cell movement in the live spleen. We show that MZ B cells are highly motile and exhibit long membrane extensions. MZ B cells shuttle between MZ and follicles with at least one fifth of the cells exchanging between compartments per hour, a behavior that explains their ability to rapidly deliver antigens from the open blood circulation to the secluded follicles. FO B cells also transit from follicles to MZ but unlike MZ B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. FO B cell egress via the MZ is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that MZ B cells migrate continually between MZ and follicles and establishes the MZ as a site of S1PR1-dependent B cell exit from follicles. The work also shows how adhesive differences of closely related cells critically influences their behavior in the same microenvironment.

The cellular dynamics of lymphocyte egress from lymph nodes are poorly defined. Here, we visualized the branched organization of lymph node cortical sinuses and found that after entry some T cells were retained while others returned to the parenchyma. Sphingosine-1-phosphate receptor 1 (S1P1)-deficient T cells probed the sinus surface but failed to enter. In some sinuses T cells became rounded and moved in a unidirectional fashion. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress where cortical sinus probing is followed by S1P1-dependent entry, capture of cells in a sinus region with flow and transport to medullary sinuses and the efferent lymph.

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