To permit adaptive immune responses against localized infections, vertebrates have evolved peripheral lymphoid organs (including the spleen, lymph nodes, and Peyer's patches) 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—and it is not possible to include all antigen specificities within each lymphoid organ. Instead, naive 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. 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 encounter between antigen-reactive cells. Precisely guided cell movement is therefore essential for antigen 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. We identified CXCL13 as a chemokine made in B cell follicles that selectively attracts B cells by engaging CXCR5. 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 (Figure 1).
B cells need to encounter intact antigen to mount an antibody response. Using two-photon microscopy, we observed the rapid delivery of opsonized (antibody-coated) antigen through the lymph to macrophages in the lymph node subcapsular sinus (Figure 2). Polyclonal B cells pick up opsonized antigens from macrophage processes and transport them to follicular dendritic cells (FDCs). Cognate B cells capture antigens from macrophage processes or from FDCs (Movie 1).
Most antibody responses depend on the interaction of antigen-specific B cells and helper T cells. We found that antigen-engaged B cells upregulate CCR7, move to the follicle–T zone boundary by chemotaxis (Figure 3), and form motile conjugates with helper T cells (Movie 2). Following receipt of T cell help, B cells move to interfollicular niches to clonally expand and some differentiate into plasma cells, while others become germinal center (GC) precursor cells and move to a supportive niche at the follicle center.
EBI2, a GPCR that responds to a newly identified oxysterol (7α,25-dihydroxycholesterol), is required for B cell positioning in interfollicular regions (Figure 3). EBI2 also promotes dendritic cell (DC) positioning in these areas. Downregulation of EBI2 is necessary for B cell movement to the follicle center and participation in the GC response. These findings raise questions about how the production and distribution of an oxysterol are regulated and whether this lipid influences additional immune response parameters, questions we are actively pursuing.
Cell egress from lymphoid organs is essential for immune function, yet how cells exit tissues has only recently come to light. 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 (S1PR1) is required for T cells to exit the thymus and for T and B cells to exit lymph nodes, Peyer's patches, and the spleen. Imaging analysis defined a multistep model of egress where lymphocytes contact and probe vascular sinuses, enter in an S1PR1-dependent fashion, and then become caught in a region of flow and commit to leaving the organ (Figure 4). This work provided insight into the mechanism of action of fingolimod, an S1PR1-modulating drug that inhibits egress and prevents effector lymphocytes reaching sites of inflammation. Fingolimod was approved by the Food and Drug Administration in 2010 as a treatment for multiple sclerosis.
During infection, lymphocyte egress is "shut down" in the responding lymphoid organs to allow accumulation of possible responder cells. Type I interferons can cause shutdown, and they inhibit lymphocyte S1P responsiveness by upregulating CD69, which physically associates with and inhibits S1PR1 (Figure 5). We are working on new molecular, cellular, and imaging methods to dissect the mechanisms promoting and controlling lymphocyte egress.
The GC (Movie 3) is a remarkable "training" environment that accepts low-affinity B cells in and lets a diversity of high-affinity cells out. Generated by rapidly dividing antigen-reactive B cells, the GC is organized into a "dark" zone of somatically mutating B cells and a "light" zone where B cells capture FDC-displayed antigen and interact with helper T cells. Organization of GC B cells into zones depends on CXCR4. We found that GC B cells transition from a dark zone to a light zone phenotype according to an intrinsic timer, but access to the dark zone was required for efficient somatic mutation. This work has raised questions that we are now pursuing about the nature of the intrinsic timer and about the stromal cells that support and organize the GC.
GC B cells are confined within the GC, and this is crucial for controlling growth of these rapidly dividing and mutating cells. GC B cells abundantly express S1PR2 (Figure 3), a receptor that couples to Gα13 and signals via Rho to inhibit cell migration. S1PR2 deficiency leads to GC B cell deconfinement. S1PR2 also regulates cell growth by dampening AKT signaling, and mice lacking S1PR2 frequently develop GCB-diffuse large B cell lymphoma (DLBCL). In collaboration with Louis Staudt (National Cancer Institute) we have identified S1PR2 function-disrupting mutations in human cases of GCB-DLBCL. We are performing new investigations to understand the extent of S1PR2-signaling pathway disruption in this lymphoma.
As a consequence of their central surveillance function, lymph nodes are a site of rapid exposure to invading pathogens. While studying subcapsular macrophages we discovered a heterogeneous population of effector lymphocytes situated close to these cells and thus to sites of antigen arrival in lymph nodes (Movie 4). We characterized a subset of these effectors as γδ T cells committed to production of the antibacterial and antifungal cytokine interleukin-17 (IL-17). This work led us to discover a closely related population of IL-17+ γδ T cells in the skin. We are now studying how dermal and lymph node γδ T cells and related intraepithelial lymphocytes in the intestine become positioned at these frontier locations and how they participate in barrier immunity.
Grants from the National Institutes of Health provided partial support for these projects.
As of July 28, 2014