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Cell Migration and Communication in the Immune System
Research Summary
Jason Cyster's laboratory studies how cells and antigens come together to generate immune responses. Cyster's group focuses on deciphering the molecular cues that guide immune cell movements and interactions within lymphoid organs, and the signals that promote cell egress from these organs. In complementary studies they address the basis for B lymphocyte selection during antibody affinity maturation in germinal centers and the requirements for barrier immunity in lymphoid organs and at epithelial surfaces.
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).
Figure 1: Lymphoid chemokine expression in mouse lymph node.
Adjacent sections were hybridized with RNA probes specific for CXCL13, CCL21, and CCL19. Signal is seen as black staining. The CXCL13 signal is localized in B cell follicles; the CCL21 and CCL19 signals are limited to the T cell area.
Figure 2: Capture of opsonized antigen by subcapsular sinus macrophages and follicular B cells.
Microscopy of draining lymph nodes collected 2 hours after phycoerythrin (PE) injection into mice passively immunized with antibody against PE. Sections were stained with anti-CD169 (green) to highlight macrophages and anti-B220 (blue) to label B cells. PE-immune complexes (ICs) are visualized as red puncta. Dashed line indicates lymph node capsule. Boxed area at left is enlarged at right. Arrow (right) indicates a macrophage process loaded with PE-ICs extending into the follicle. Scale bars, 20 µm.
Figure 3: Cues guiding B cell movements during the early antibody response.
Each panel shows a follicle (light and dark blue shading) and part of the adjacent T zone. Prior to antigen encounter (day 0), naive B cells migrate over follicular dendritic cell (FDC) processes throughout the follicle in a CXCL13-CXCR5–dependent manner. Antigen encounter causes B cells to upregulate CCR7 and travel to the T zone in response to CCL21 (light brown shading) for interaction with helper T cells (day 1). Helper T cells (not shown) trigger sustained EBI2 and reduced CCR7 expression, and the activated B cells move to outer- and interfollicular regions (day 2–3) in response to 7α,25-dihydroxycholesterol (dark blue shading). Some activated B cells then downregulate EBI2 and upregulate the migration inhibitory S1PR2 receptor and move away from regions suggested to be high for S1P (contours) to begin a germinal center reaction inside the follicle (day 4+).
Jason Cyster
Figure 4: Multistep model of lymph node egress.
Step 1: Randomly migrating T cell (green) makes contact with and probes a cortical (lymphatic) sinus. Step 2: Probing T cell undergoes S1PR1-dependent commitment to enter sinus. Step 3: T cell detaches from sinus wall, rounds up, and becomes caught in a region of lymph flow that carries the cell to the medullary sinuses and efferent lymphatic. Cortical sinuses begin as branched structures in the T zone, often near high endothelial venules (HEVs), and connect to macrophage-containing medullary sinuses and the efferent lymphatic.
Figure 5: Model of CD69-mediated block in egress. Innate immune stimuli induce type I interferons (e.g., IFNα/β), which engage receptors on naïve lymphocytes, leading to transcriptional up-regulation of CD69. CD69 protein physically associates with and inhibits S1P1 function and down-regulates the receptor. Inhibition of lymphocyte S1P1 prevents transmission of the egress-promoting signal, causing lymphocyte retention in the responding lymphoid organ.
Jason Cyster
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).
Movie 1: Real-time imaging of antigen capture by cognate B cells from follicular dendritic cells (FDCs). Two time-lapse image sequences of 20 μm z projections from inguinal lymph node explants showing acquisition of antigen (HEL-PE, red) by green-labeled antigen-specific (cognate, MD4) B cells from FDC processes. In both examples, cognate B cells capture large aggregates of antigen (arrowhead). Some noncognate CFP-transgenic B cells are observed in cyan. Elapsed time is shown as hours:minutes:seconds.
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.
Movie 2: Time-lapse image sequence shows cognate interactions between antigen-specific B cells (red) and T cells (green) within a responding lymph node ~30 hours after challenge with antigen in adjuvant. Time indicated as h:m:s. Each image is 135 x 114 µm and projects a depth of 51 µm.
From Okada, T., Miller, M.J., Parker, I., Krummel, M.F., Neighbors, M., Hartley, S.B., O'Garra, A., Cahalan, M.D., and Cyster, J.G. 2005. PLoS Biology Jun;3(6):e150, video S6.
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.
Movie 3: Intravital imaging of germinal center (GC) B cells. A time-lapse sequence of 21-µm z-projection images of a mouse inguinal lymph node GC seven days after immunization. Approximately three percent total GC B cells are labelled with green fluorescent protein and a similar fraction of naive B and T cells are rhodamine labelled (red). Cell division and death are annotated in the video as well as a moving bleb from an apoptotic cell (most likely carried by a migrating T cell). Also indicated is a blood vessel in which four labeled cells (arrowheads) appeared in the image stack within 20 s and disappeared within the next 20 s. Elapsed time is shown as h:m:s.
From Allen, C.D., Okada, T., Tang, H.L., and Cyster, J.G. Science 2007 315:528–531
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.
Movie 4: Two-photon microscopy of effector lymphocytes surveying subcapsular sinus macrophages in the lymph node. Intravital two-photon laser scanning microscopy showing CXCR6hi effector lymphocytes (green) migrating in close association with CD169+ subcapsular sinus macrophages (red) in an explanted Cxcr6GFP/+ mouse lymph node. Cyan+ cells are CFP-transgenic B cells that were transferred to the mouse the day before and make up about 1 percent of total B cells. The dark blue appearance of the lymph node capsule is due to second harmonic resonance of collagen. Movie shows 12 µm maximum intensity z projection. Time is shown as hours:minutes:seconds. FO, follicle; SCS, subcapsular sinus.
From Gray, E.E., Friend, S., Suzuki, K., Phan, T.G., and Cyster J.G. 2012. PLoS One 7, e38258.
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.
