Multiple sclerosis is regarded as a prototypical disease state resultant of neuroinflammation. RTLs have been engineered to augment antigen-driven immunosuppression of autoreactive T cells by presenting TCR ligands (self-antigen plus MHC molecule) in the absence of co-stimulatory signals that are normally supplemented by antigen presenting cells (APCs). RTLs have been shown to reduce T cell-driven encephalitogenicity and both clinical and histological signs of EAE. Previous studies suggested that RTLs confer a therapeutic benefit via the downregulation of T cell-mediated inflammation and by modifying the permeability of the blood-brain barrier in a murine model of EAE or ischemic stroke. RTLs were found to decrease the levels of systemic chemokines and adhesion molecules on the CNS endothelium as well as cytokines that switch on anti-inflammatory effectors rather than increasing antibody production by B cells. Although RTLs were originally thought to act as a partial agonist via TCR, our recent study demonstrated the inability of splenic CD3+ T cells to bind RTLs. Rather, we found that RTLs bound APC populations including B cells, macrophages, and dendritic cells, in an antigenic-independent manner. Perhaps RTL-armed splenic APCs suppress the transfer of EAE by antigen-stimulated T cells. Alternatively, peripheral blood cells may bind RTL through an unknown surface receptor distinct from TCR, indirectly inducing T cell tolerance against self-antigen. Our study was designed to determine whether peripheral blood cell populations, such as platelets, can bind to RTL, thus offering a level of redundancy to APCs.
Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing an essential role in inflammation and immunity[5, 7]. Activated platelets shed granules and microparticles which contain a variety of adhesive molecules and immunomodulatory factors, resulting in the localized recruitment of immune cells under shear. Abnormal platelet activation (microparticle presence, P-selectin expression, and increased circulating aggregates) has been documented in MS patients. A recent gene microarray study revealed that the platelet αIIbβ3 receptor was transcriptionally upregulated in MS chronic lesions. Perhaps RTL may modulate the pathophysiological role that platelets play in neurodegenerative diseases such as MS.
A number of platelet ligands and pathways have been identified that negatively regulate platelet function. For instance, recent studies have shown negative regulatory roles for the platelet ligands semaphorin 3A, histone H1, thrombospondin-1, and low-density lipoprotein. Our current study provides evidence that RTL, in addition to serving as a platelet ligand, may serve to negatively regulate agonist-induced platelet function, as evidenced by the fact that RTL down-regulated platelet aggregation to collagen. Moreover, our study provides evidence that RTL inhibits occlusive thrombus formation on collagen-coated surfaces under physiologically relevant pressure gradients.
Among various platelet surface RTL-receptors, one potential candidate we identified was CD40, a co-stimulatory molecule that regulates lymphocyte signaling upon antigen presentation. CD40 is known to contribute to the initiation, activation, and amplification of immune responses. CD40-CD40 ligand (CD40L, or CD154) interactions are well described in MS, as CD40L+ T cells are known to be greatly increased in MS patients[29, 30]. Moreover, CD40 is expressed on platelets and APCs, but not T cells, in accord with the peripheral blood populations we have shown to bind RTL. We therefore tested the hypothesis that CD40 was the platelet receptor for RTL. Our preliminary findings demonstrated that purified platelets from CD40-/- mice bind to RTL at the same level as compared to platelets from wild type mice (unpublished observations, AI and OJTM), arguing against a role for CD40 as an RTL-receptor on mouse platelets. Our future efforts will be focused on identifying the putative RTL-receptor on peripheral blood platelets. The characterization of the molecular mechanisms by which RTL binds to peripheral blood cells may better the understanding of the pharmacokinetics of RTL drug delivery as well as the further development of RTL for therapy in autoimmune diseases.