Optical tomographic imaging of near infrared imaging agents quantifies disease severity and immunomodulation of experimental autoimmune encephalomyelitis in vivo.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model that captures many of the hallmarks of human multiple sclerosis (MS), including blood-brain barrier (BBB) breakdown, inflammation, demyelination and axonal destruction. The standard clinical score measurement of disease severity and progression assesses functional changes in animal mobility; however, it does not offer information regarding the underlying pathophysiology of the disease in real time. The purpose of this study was to apply a novel optical imaging technique that offers the advantage of rapid imaging of relevant biomarkers in live animals. METHODS: Advances in non-invasive fluorescence molecular tomographic (FMT) imaging, in combination with a variety of biological imaging agents, offer a unique, sensitive and quantifiable approach to assessing disease biology in living animals. Using vascular (AngioSense 750EX) and protease-activatable cathepsin B (Cat B 680 FAST) near infrared (NIR) fluorescence imaging agents to detect BBB breakdown and inflammation, respectively, we quantified brain and spinal cord changes in mice with relapsing-remitting PLP139-151-induced EAE and in response to tolerogenic therapy. RESULTS: FMT imaging and analysis techniques were carefully characterized and non-invasive imaging results corroborated by both ex vivo tissue imaging and comparison to clinical score results and histopathological analysis of CNS tissue. FMT imaging showed clear differences between control and diseased mice, and immune tolerance induction by antigen-coupled PLGA nanoparticles effectively blocked both disease induction and accumulation of imaging agents in the brain and spinal cord. CONCLUSIONS: Cat B 680 FAST and AngioSense 750EX offered the combination best able to detect disease in both the brain and spinal cord, as well as the downregulation of disease by antigen-specific tolerance. Non-invasive optical tomographic imaging thus offers a unique approach to monitoring neuroinflammatory disease and therapeutic intervention in living mice with EAE.

Induction of transplantation tolerance by allogeneic donor-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Several studies have shown that recipient-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are involved in transplantation tolerance. However, it is not clear whether allogeneic donor-derived Tregs are able to regulate T cell alloreactivity after solid organ allograft transplantation. Related studies in experimental bone marrow transplantation have shown that allogeneic donor-derived Tregs are capable of promoting early and long-term allogeneic hematopoietic engraftment, accompanied by tolerance to donor and recipient antigens. However, in these models, donor-derived Tregs are syngeneic with respect to the T responder cells. The role of Tregs in solid organ transplantation models where recipient-derived T responder and donor-derived Tregs are allogeneic has been scarcely studied. In order to determine whether allogeneic Tregs were able to regulate T cell alloreactivity, CD4(+)CD25(-) and CD8(+) T responder cells were cultured with stimulator dendritic cells in several responder-stimulator strain combinations (C57BL/6-->BALB/c, BALB/c-->C57BL/6 and C3H-->BALB/c) in the presence of responder-derived, stimulator-derived or 3rd-party-derived Tregs. Then, the frequency of IFN-gamma+ alloreactive T cells was determined by means of ELISPOT assay. The results of this study demonstrate that, regardless of the responder-stimulator strain combination, both responder-derived and stimulator-derived Tregs, but not 3rd-party-derived Tregs, significantly inhibited CD4(+) and CD8(+) T cell alloreactivity. The effect of allogeneic stimulator-derived Tregs was dependent on IL-10 and TGF-beta and reversed by exogenous IL-2. In vivo experiments in nu/nu recipients reconstituted with CD4(+)CD25(-) T responder and Tregs showed that recipient and donor-derived, but not 3rd-party-derived Tregs, significantly enhanced skin allograft survival. Importantly, T cells from both recipient-derived and donor-derived Treg-reconstituted nu/nu recipients exhibited donor-specific unresponsiveness in vitro. These results show that allogeneic donor-derived Tregs significantly inhibit T cell alloreactivity and suggest their potential use in the induction of transplantation tolerance.