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 immune 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. This research challenge is strikingly exemplified 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. There is also evidence that the success of current immunotherapies for cancer is tightly correlated with the efficiency of immune cell access to the tumor microenvironment.

Major Goals:

• Decipher the guidance cue codes controlling leukocyte migration and interaction events during tissue surveillance and immune response
• Visualize immune response dynamics using advanced imaging and cell engineering approaches
• Define the selection mechanisms for antibody affinity maturation and those that help prevent autoantibody production
• Characterize the cellular dynamics underlying mucosal immune responses
  

On-going Research:

Chemokines and Lipid Mediators 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. More recently we identified a role for an intercellular signaling lipid, the oxysterol 7a,25-dihydroxycholesterol, in guiding B cell, dendritic cell (DC) and T follicular helper (Tfh) cell movements and in supporting humoral immune responses (Fig. 1). We have also determined how the oxysterol 25-hydroxycholesterol controls IL1-family cytokine production by macrophages. An important thrust of our ongoing work is a discovery program to identify unaccounted for extracellular cues that control multiple other immune cell migration, interaction and communication processes.

Antigen Encounter Dynamics: Using real-time 2-photon microscopy we have visualized the dynamics of B cell–antigen encounters in intact lymphoid organs (see Video Gallery); this work highlighted new roles for sinus-lining macrophages and follicular dendritic cells (FDCs). The properties of stromal cells that facilitate their functions in antigen display and as niche organizers are under continued investigation using approaches such as scRNAseq (Fig. 2) and knockout mouse studies. We also investigate requirements for DC homeostasis and antigen encounter. We demonstrated that splenic cDC2s position in blood-exposed locations in an EBI2-dependent manner and identified a novel mechanism for their activation by missing self-CD47 detection.

Cell Egress from Tissues: Lymphocyte egress from lymphoid tissues into circulation is necessary for them to carry out their effector functions, whether that is to continue their surveillance program, to kill infected cells or tumor cells, or to drive chronic inflammatory disease. We have shown that the blood lipid, sphingosine-1-phosphate (S1P), acts via S1PR1 to promote egress from peripheral lymphoid organs and thymus. Our work helped define the mode of action of a compound, FTY720 (Fingolimod), that inhibits lymphocyte egress and is now approved for treatment of several autoimmune diseases. We also found that the lymphocyte activation antigen, CD69 acts as a physiological egress regulator by inhibiting S1PR1 function (Fig. 3). Recently, we demonstrated that a second S1P receptor, S1PR2, acts with CD69 to counter S1PR1 function and promote tissue residence of T cells. In contrast to chemoattractant receptors that couple via Gai, S1PR2 couples to Ga13 and mediates migration-inhibition. Future work will test the role of this and other pathways in promoting tissue retention of further immune cell types, such as T resident memory (Trm) cells. We will continue to use cell engineering approaches to test the sufficiency of molecules to guide immune cell migration and function in vivo.

Affinity Maturation and Germinal Center Responses: Although first identified in the 1800’s, the inner workings of the germinal center – sites of antibody diversification and affinity maturation – are still incompletely understood. We found a role for chemokines in organizing the structure into light and dark zones and for S1P and S1PR2 in promoting niche confinement and growth control. By real-time imaging we have begun to characterize B and T cell migration and interaction dynamics during these responses and we are using new signaling reporters to study the signals underlying selection. Using classical biochemistry and mass spectrometry approaches we identified a novel metabolite, S-geranylgeranyl-L-glutathione (Ggg) as a ligand for P2RY8, a receptor that functions together with S1PR2 in promoting germinal center cell confinement in humans (Fig. 4). In collaborative studies, we showed how loss of S1PR2, P2RY8 or the downstream signaling protein, Ga13, contributes to the development of germinal center-derived lymphomas. We are using biochemical approaches to study Ggg synthesis and transport, as well as genetic approaches to define the physiological roles of P2RY8 and Ggg.

Ongoing studies combine perturbations in communication molecules (such as HVEM-BTLA) with methods to measure affinity maturation, including scV(D)Jseq, and computational approaches to further define how selection of high affinity clones - and elimination of low affinity and autoreactive clones - occurs. We are also using CRISPR/Cas9-based in vivo screening to define requirements for the formation of long-lived memory B cells and plasma cells. In future work, we will study the basis for the apparent lack of long-lived humoral immune responses following coronavirus infections.

Mucosal Immunity: IgA is the major antibody isotype produced in the body, yet the mechanism of mucosal B cell encounter with nasal or 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 respiratory and intestinal pathogens. We are also interested in how immune cells survey epithelial surfaces and contribute to tissue homeostasis. This includes work on EBI2 function in the lung and GPR18 and GPR55 function in the gut. We also study the function of IL17-producing gdT cells in the skin, and this includes a growing interest in neuro-immune cross-talk in this barrier tissue.