Charlotte M. Vines

Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ± 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ± 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2−/−/arrestin 3−/− murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.

A novel IL-17 signaling pathway controlling keratinocyte proliferation and tumorigenesis via the TRAF4-ERK5 axis.

Wu et al. report a novel IL-17–mediated cascade via the IL-17R–TRAF4–ERK5 axis that directly stimulates keratinocyte proliferation and skin tumor formation in mice.

Codelivery of antigen and an immune cell adhesion inhibitor is necessary for efficacy of soluble antigen arrays in experimental autoimmune encephalomyelitis

Autoimmune diseases such as multiple sclerosis (MS) are typified by the misrecognition of self-antigen and the clonal expansion of autoreactive T cells. Antigen-specific immunotherapies (antigen-SITs) have long been explored as a means to desensitize patients to offending self-antigen(s) with the potential to retolerize the immune response. Soluble antigen arrays (SAgAs) are composed of hyaluronic acid (HA) cografted with disease-specific autoantigen (proteolipid protein peptide) and an ICAM-1 inhibitor peptide (LABL). SAgAs were designed as an antigen-SIT that codeliver peptides to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Codelivery of antigen and cell adhesion inhibitor (LABL) conjugated to HA was essential for SAgA treatment of EAE. Individual SAgA components or mixtures thereof reduced proinflammatory cytokines in cultured splenocytes from EAE mice; however, these treatments showed minimal to no in vivo therapeutic effect in EAE mice. Thus, carriers that codeliver antigen and a secondary “context” signal (e.g., LABL) in vivo may be an important design criteria to consider when designing antigen-SIT for autoimmune therapy.

Nanoparticles targeting dendritic cell surface molecules effectively block T cell conjugation and shift response.

Dendritic cells (DCs) are potent professional antigen presenting cells (APC) that activate naïve T cells. Interaction of ICAM-1 and LFA-1 molecules on each cell is required for T cell conjugation to DCs, which leads to naïve CD4+ T cell activation and proliferation. Nanoparticles capable of blocking LFA-1/ICAM-1 interaction were studied as inhibitors of T cell conjugation to DCs. Primary DCs were primed with ovalbumin, then treated with a peptide that binds ICAM-1 (LABL), a peptide that binds LFA-1 (cIBR), or the same peptides covalently linked to the surface of poly(dl-lactic-co-glycolic acid) nanoparticles (NPs). LABL-NPs and cIBR-NPs rapidly bound to DCs and inhibited T cell conjugation to DCs to a greater extent than the free peptides, unconjugated nanoparticles (NPs), anti-ICAM-1 antibodies, and anti-LFA-1 antibodies. In addition, DCs treated with NPs or with cIBR-NPs stimulated the proliferation of T cells, but DCs treated with LABL-NPs did not stimulate T cell proliferation. Nanoparticles targeting ICAM-1 or LFA-1 also altered cytokine production by DC cocultured with T cells when compared to free ligands, suggesting that these NPs may offer a unique tool for shaping T cell response.

Co-Delivery of Autoantigen and B7 Pathway Modulators Suppresses Experimental Autoimmune Encephalomyelitis

Autoimmune diseases such as multiple sclerosis (MS) are characterized by the breakdown of immune tolerance to autoantigens. Targeting surface receptors on immune cells offers a unique strategy for reprogramming immune responses in autoimmune diseases. The B7 signaling pathway was targeted using adaptations of soluble antigen array (SAgA) technology achieved by covalently linking B7-binding peptides and disease causing autoantigen (proteolipid peptide (PLP)) to hyaluronic acid (HA). We hypothesized that co-delivery of a B7-binding peptide and autoantigen would suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Three independent B7-targeted SAgAs were created containing peptides to either inhibit or potentially stimulate the B7 signaling pathway. Surprisingly, all SAgAs were found to suppress EAE disease symptoms. Altered cytokine expression was observed in primary splenocytes isolated from SAgA-treated mice, indicating that SAgAs with different B7-binding peptides may suppress EAE through different immunological mechanisms. This antigen-specific immunotherapy using SAgAs can successfully suppress EAE through co-delivery of autoantigen and peptides targeting with the B7 signaling pathway.

Autoimmune therapies targeting costimulation and emerging trends in multivalent therapeutics

Proteins participating in immunological signaling have emerged as important targets for controlling the immune response. A multitude of receptor-ligand pairs that regulate signaling pathways of the immune response have been identified. In the complex milieu of immune signaling, therapeutic agents targeting mediators of cellular signaling often either activate an inflammatory immune response or induce tolerance. This review is primarily focused on therapeutics that inhibit the inflammatory immune response by targeting membrane-bound proteins regulating costimulation or mediating immune-cell adhesion. Many of these signals participate in larger, organized structures such as the immunological synapse. Receptor clustering and arrangement into organized structures is also reviewed and emerging trends implicating a potential role for multivalent therapeutics is posited.

Hyaluronic acid graft polymers displaying peptide antigen modulate dendritic cell response in vitro.

A novel oxime grafting scheme was utilized to conjugate an ICAM-1 ligand (LABL), a cellular antigen ovalbumin (OVA), or both peptides simultaneously to hyaluronic acid (HA). Samples of HA only and the various peptide grafted HA were found to bind to dendritic cells (DCs). HA with grafted LABL and OVA showed the greatest binding to DCs. Dendritic cells treated with HA, HA with grafted LABL, or HA with grafted LABL and OVA significantly suppressed T cell and DC conjugate formation and T cell proliferation and reduced proinflammatory cytokine production compared to untreated cells. These results suggest that HA serves as an effective backbone for multivalent ligand presentation for inhibiting T cell response to antigen presentation. In addition, multivalent display of both antigen and an ICAM-1 inhibitor (LABL) may enhance binding to DCs and could potentially disrupt cellular signaling leading to autoimmunity.

Phospholipase C Isoform Functions in Immune Cells

Phospholipase C (PLC) family members play critical roles in regulating immune cell functions during inflammatory responses. This chapter discusses how different family members can be activated by G-protein coupled receptors, T-cell receptors, B-cell receptors, and other tyrosine kinase receptors, in addition to many of the pathways that contribute to propagation of signaling through the intracellular signaling events that are mediated by different family members. By understanding these signaling events and immune mechanisms we will be able to better define targets for pharmacological intervention for inflammation and autoimmune diseases.