Cell Migration Dynamics and Intercellular Communications Underlying Immunity
Background: The immune system is composed of multiple cell types that are distributed in lymphoid and non-lymphoid tissues throughout the body. Understanding how these cells help maintain tissues in a healthy metabolic state and ensure homeostasis with commensals while being able to respond rapidly to foreign invaders or malignant self requires a precise understanding of how they position in tissues, and how they communicate. A striking example of this challenge occurs during humoral immune responses, where rare antigen-specific B and T lymphocytes must first encounter antigen and then interact with each other to mount an antibody response. Another example is the continual surveillance of epithelial surfaces by innate lymphocytes. The importance of understanding immune cell trafficking is also highlighted by evidence that the success of tumor immunotherapy is tightly correlated with the efficiency of immune cell access to the tumor microenvironment.
Major Goals: (i) Decipher the guidance cue codes controlling leukocyte migration and interaction events during tissue surveillance and immune responses; (ii) Visualize immune response dynamics using advanced imaging approaches; (iii) Define the selection mechanisms for antibody affinity maturation and that help prevent autoantibody production; (iv) Characterize requirements for mounting mucosal immune responses.
Chemokines and Oxysterols as Tissue Organizers: Chemokines are small secreted chemoattractive proteins that signal via heterotrimeric G-protein coupled receptors (GPCRs). We have demonstrated that several chemokines are expressed in lymphoid organs and function in guiding lymphocyte migration. Recently we identified a role for an intercellular signaling lipid, the oxysterol 7a,25-dihydroxycholesterol (7a,25-HC), in guiding B cell, dendritic cell (DC) and T follicular helper (Tfh) cell movements and supporting humoral immune responses. Efforts are ongoing to understand how production of this oxysterol is regulated and what additional influences it has on immune function. This includes a new branch of work aimed at defining how 25-hydroxycholesterol controls IL1-family cytokine production by macrophages. We also continue a discovery program to identify unaccounted for cues that control multiple other immune cell migration and interaction processes.
Figure 1. Cues guiding antigen-surveillance dynamics of B lymphocytes and their migration pattern in the lymph node. From Cyster 2010 Nature Immunology 11, 989-96.
Antigen Encounter Dynamics: Using real-time 2-photon microscopy we have visualized the dynamics of B cell–antigen encounters in intact lymphoid organs. This work highlighted new roles for sinus-lining macrophages and follicular dendritic cells (FDCs). The properties of these accessory cells that facilitate their roles in antigen display and as niche organizers are under continued investigation using approaches such as single cell RNAseq and reporter mouse construction.
Figure 2. Tracks (~60 min duration) of antigen-specific B cells migrating within a lymph node follicle before (naïve) and 2 hrs after vaccination with the cognate antigen. Circles indicate end of tracks. From Okada, Miller et al., 2005 Plos Biology 3, e150.
Cell Egress from Tissues: A lot is known about how cells get into lymphoid tissues from blood, but less is understood about how they get out. Yet they must get out to mediate their effector function at sites of infection or autoimmune inflammation. We have shown that the blood lipid, sphingosine-1-phosphate (S1P), is involved. Like chemokines, S1P signals via GPCRs, and we found that if lymphocytes lack one of the five S1P receptors, S1PR1, they are unable to leave the thymus or peripheral lymphoid organs. Our studies helped define the mode of action of a compound, FTY720 (Fingolimod) that inhibits lymphocyte egress and has been approved for treatment of multiple sclerosis. We also found that the lymphocyte activation antigen, CD69 acts as a physiological egress regulator by inhibiting S1PR1 function. Further studies, involving CRISPR/Cas9-based screens and cell biological and real-time imaging approaches, are aimed at defining further requirements for cell egress from tissues into circulation.
Figure 3. Innate stimuli cause lymph node egress shutdown by induction of CD69, an inhibitor of S1PR1. From Cyster and Schwab 2012 Annual Reviews of Immunology 30, 69-94.
Affinity Maturation and Germinal Center Responses: Although first identified in the 1800's, the inner workings of the germinal center - the site of antibody affinity maturation - are still incompletely understood. We found a role for chemokines in organizing the structure into light and dark zones and for S1P, acting via the migration inhibitory S1PR2 receptor, in promoting niche confinement and growth control. Using real-time imaging we have begun to characterize B and T cell migration and interaction dynamics during the selection events associated with this tightly regulated response. Ongoing studies combine perturbations in organizing molecules with methods to measure affinity maturation and imaging approaches to further define how selection of high affinity clones - and elimination of low affinity and autoreactive clones - occurs. We are also aiming to apply our developing knowledge about B cell response dynamics to improving vaccine design, focusing on influenza antigens.
Figure 4. Germinal center B cells transition between light and dark zone states according to Tfh cell inputs and an intrinsic timer. From Bannard et al., 2013 Immunity 39, 912-24.
Mucosal Immunity: IgA is the major antibody isotype produced in the body, yet the mechanism of mucosal B cell encounter with gut antigens and the factors controlling IgA isotype switching are incompletely understood. We are applying approaches used to study splenic and lymph node B cell responses to characterize the requirements for mounting IgA responses against commensal flora and intestinal pathogens. We are also interested in how immune cells survey epithelial surfaces. We are following up on our discovery that an orphan receptor, GPR18, is needed for intraepithelial lymphocyte (IEL) accumulation in the small intestine with studies to define how this receptor and others promote the surveillance function.
Figure 5. Migration cues and cell-cell interaction requirements for induction of IgA isotype switching in Peyer’s patches. From Reboldi et al., 2016 Science 352, aaf4822.