During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seventransmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1͞CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1␤͞ CCL4, and RANTES͞CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

Lymphotoxin beta receptor (LTbetaR)-/- mice were created by gene targeting. LTbetaR-/- mice lacked Peyer's patches, colon-associated lymphoid tissues, and all lymph nodes. Mucosa patrolling alphaEbeta7high integrin+ T cells were virtually absent. Spleens lost marginal zones; T/B cell segregation and follicular dendritic cell networks were absent. Peanut agglutinin+ cells were aberrantly detectable around central arterioles. In contrast to TNF receptor p55-/- mice, antibody affinity maturation was impaired. Since LTbetaR-/- mice exhibit distinct defects when compared to LTalpha-/- and LTbeta-/- mice, it is suggested that the LTbetaR integrates signals from other TNF family members. Thus, the LTbetaR proves pivotal for the ontogeny of the secondary lymphoid tissues. Furthermore, affinity maturation is dependent on LTalpha1beta2 rather than on LTalpha3.

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.