To permit adaptive immune responses against localized infections, vertebrates have evolved peripheral lymphoid organs (including the spleen and lymph nodes) that filter and concentrate molecules (antigens) from the nearby tissue and then display them to lymphocytes. The frequency of lymphocytes specific for any given pathogen is low—perhaps 1 per 100,000 lymphocytes—and it is not possible to include all lymphocyte specificities within each lymphoid organ. Instead, naïve lymphocytes travel continuously between different lymphoid organs, surveying for their specific antigen.
After entering a lymphoid organ from the blood, B cells move to the lymphoid follicle, while T cells localize in an adjacent T zone (Figure 1). Lymphocytes spend about a day surveying antigen-presenting cells within their microenvironment for antigen before returning to circulation. If antigen is encountered, the cells undergo striking changes in migration, stopping within the organ and moving to locations that favor encounters between antigen-reactive cells (Figure 2). Precisely regulated cell movement is therefore essential for immune surveillance and for mounting immune responses.
The primary organizers of lymphoid tissues are chemokines, secreted proteins that act via G protein–coupled receptors (GPCRs) to promote chemotactic migration of cells. CXCL13 is a chemokine that is made in the B cell follicles and selectively attracts B cells. Using gene targeting in mice, we demonstrated that CXCL13 is necessary for B cell localization within follicles. Two chemokines, CCL19 and CCL21, that are ligands for CCR7 are expressed by stromal cells in the T zone and attract T cells into this compartment.
B cells need to encounter intact antigen to mount an antibody response, yet the dynamics of this encounter have been poorly understood. Using real-time two-photon microscopy, we observed the rapid delivery of antigen in the form of immune complexes through the lymph to macrophages in the lymph node subcapsular sinus. B cells pick up immune complexes from macrophage processes that penetrate the follicle and transport them to follicular dendritic cells (FDCs). Cognate B cells capture antigens from macrophage processes or from FDCs. Ongoing studies are characterizing how subcapsular macrophages and FDCs display antigens and interact with B cells.
Most antibody responses depend on the interaction of antigen-specific B cells and helper T cells. By two-photon microscopy we showed that antigen-engaged B cells move to the follicle–T zone boundary by chemotaxis up a CCR7 ligand gradient. Many cognate interactions between B cells and helper T cells last 10–40 minutes, and some persist for >1 hour (Movie 1), whereas noncognate interactions last less than 10 minutes. We found that B cells responding to T cell help up-regulate EBI2 and are guided by this orphan GPCR to a newly defined niche in the outer follicle. Down-regulation of EBI2 is necessary for movement to the follicle center and participation in the germinal center (GC) response.
The GC is a remarkable "training" environment that accepts low-affinity B cells in and lets only high-affinity cells out. How this microenvironment supports selection of high-affinity B cells remains unclear. Generated by rapidly dividing antigen-reactive B cells, it forms over about a week and becomes organized into a "dark" zone of somatically mutating B cells and a "light" zone where B cells compete for FDC-displayed antigen. We found that organization of GC B cells into light and dark zones depends on the chemokine receptor CXCR4. GC B cells migrate extensively within the FDC network and undergo cell division and cell death in both light and dark zones (Movie 2). Long interactions with helper T cells are rare. These studies suggest the need for a revised model of selection events within the GC, where cells compete not only for antigen but also for T cell help. Studies are under way to test this model.
Cell egress from bone marrow, thymus, and peripheral lymphoid organs is necessary for immune function. While much has been defined regarding how cells get into tissues from blood, little has been understood about how they get out. Sphingosine-1-phosphate (S1P), a lipid present in plasma and lymph, acts as a signaling molecule by engaging any of five GPCRs. We found that S1P receptor-1 (S1P1) is highly expressed in lymphocytes and that it is required for T cells to exit the thymus and for T and B cells to exit lymph nodes and spleen. Following up on toxicology studies that had suggested a caramel food colorant could inhibit thymic egress, we discovered that the active compound inhibited S1P lyase and disrupted the S1P gradient between tissue and circulatory fluid. Several small-molecule inhibitors of S1P lyase are now being developed in industry as possible therapeutics for treatment of autoimmune diseases.
To define the source of S1P needed for lymphocyte egress we collaborated with Shaun Coughlin (University of California, San Francisco) in a study of mice conditionally ablated for the two S1P-synthesizing sphingosine kinases. Mice were generated that lacked circulating S1P, and lymphocyte egress in these animals was blocked. Using intravital microscopy, we defined a multistep model of lymphocyte egress from lymph nodes where lymphocytes contact and probe lymphatic sinuses, enter in an S1P1-dependent fashion, and then become caught in a region of flow and commit to exiting the organ (Figure 3). Ongoing imaging and cell biological studies aim to define the nature of the S1P1-mediated egress-promoting signal.
Defects in lymphocyte egress can be associated with immunodeficiency. By genetic mapping studies in a spontaneous mouse mutant with a thymic egress defect we established a role for Coronin-1A, a regulator of actin branching, in T cell egress. This work led us to collaborate with Jennifer Puck (University of California, San Francisco) and identify a case of human SCID (severe combined immunodeficiency) caused by a loss-of-function Coronin-1A mutation. This represents the first example of human immunodeficiency associated with an egress defect. A screen of mice harboring ethylnitrosourea-induced mutations for lymphocyte-trafficking defects, performed in collaboration with Christopher Goodnow (Australian National University), led us to identify S1P5 as a T-bet–induced gene needed for natural killer (NK) cell egress from bone marrow and lymph nodes. Other mutant mouse lines identified as having altered circulating lymphocyte populations are undergoing characterization.
During infection, lymphocyte egress is transiently "shut down" in the responding lymphoid organs to allow accumulation of possible responder cells. Type I interferons can cause shutdown, and we found that they inhibit lymphocyte S1P responsiveness by up-regulating CD69, which physically associates with and inhibits S1P1(Figure 4). We are investigating the mechanism for this unusual mode of GPCR regulation.
The immune system is constantly challenged to maintain the most diverse possible repertoire of lymphocytes for combating foreign pathogens while at the same time minimizing the number of strongly autoreactive cells in the repertoire. We are studying self-tolerance checkpoints that help ensure our immune system does not attack self-tissue. Although many autoreactive B cells are eliminated at their site of development in the bone marrow, some cells reach circulation and need to be regulated by peripheral mechanisms. Using an immunoglobulin (Ig)-transgenic mouse model, we determined that efficient elimination of autoreactive B cells depends on competition with nonautoreactive cells. We have shown that this occurs in part through competition for the B cell survival factor, BAFF. In future studies we will attempt to define additional self-tolerance checkpoints acting on autoreactive B cells.
Grants from the National Institutes of Health provided support for these projects.
As of February 05, 2010