1. The spleen, not the lung, is the major organ where T-cells acquire pathogenicity This study, dealing with identification of the organ where T-cells acquire pathogenicity, was initiated in FY 2013, extended in FY 2014 and completed in FY 2015. In the experimental system used for this ongoing study, mouse T-cells specific against hen egg lysozyme (HEL) are transferred into mice in which HEL is transgenically expressed in the eyes. When activated, the transferred cells induce ocular inflammation, that is detected after a latent period of 2-3 days post cell transfer. This delay of disease onset is attributed to the need for a period of licensing for pathogenicity during which the transferred cells acquire the capacity to induce inflammation. A Nature paper by Odoardi et al., (2012, 488:675), concluded that the lung is THE licensing organ for pathogenic T-lymphocytes. Our previous studies provided evidence to indicate that this conclusion is incorrect: the spleen is THE organ where the majority of transferred cells are being licensed for pathogenicity. In our previous experiments we administered the adoptively transferred cells using the route employed by Odoardi et al., namely, intravenously (i.v.). The majority of cells injected by this route enter the recipient mostly via the lung, thus providing a non-physiological early accumulation of donor cells in the lung, despite the majority of donor cells migrating from the lung to other organs. To examine this issue, we carried out in FY 2015 experiments in which the donor cells were administered by other two routes: (i) donor cells activated in vitro and injected intraperitoneally (i.p.) and (ii) CD4 donor cells transferred at the nave stage and activated in vivo, in the recipient mice (by the antigen, HEL, and TLR ligands). Ocular inflammation was induced in recipients of cells administered by either one of the two procedures. Importantly, unlike in recipient mice injected i.v. with activated donor cells, we found that the donor cells in the recipient groups of the two new modes of cell transfer accumulated in the lung and spleen of the mice by the same kinetics. Moreover, the numbers of donor cells accumulating in the recipient lungs of these two new mouse groups, were found to be 100 times smaller than those in the spleens. Significantly, the number of donor cells in the lung of mice injected i.v. with activated donor cells was only 10 times smaller than that in the recipient spleen. These new data thus further support our assertion, that the conclusion of the mentioned Nature paper (Odoardi et al., 2012) is inaccurate: the spleen, not the lung is THE organ where licensing for pathogenicity takes place. Furthermore, our new data indicate that the accumulation of donor cells in the lung in the cited study is just the outcome of the mode of cell transfer, by the i.v. route. 2. Inhibition of EAU and related immune responses by immunosuppressive agents. During FY 2015 we extended the study concerning the activities of immunosuppressive compounds by testing the activities of a new compound, TMP778. Similar to Digoxin, an agent we investigated in FY 2014, TMP778 selectively inhibits the activity of ROR-gt, the transcription factor for the generation of Th17 cells. TMP778 was provided to us by Tempero Pharmaceuticals-GlaxoSmithKlinea, the company that developed this agent. Unlike Digoxin, TMP778 is non toxic to retinal cells and thus, it could be considered for treatment of uveitis in humans. TMP778 inhibited EAU development. Analysis of the suppressive activity of TMP778 and Digoxin on immune responses revealed the unexpected phenomenon that whereas these agents selectively inhibit IL-17 production by cultured T-lymphocytes, spleen cells of mice treated with these agents exhibited stronger reduction of interferon-gamma (the signature cytokine for Th1 cells) than of IL-17. Analysis of this phenomenon is under way.
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