NEW INFORMATION ON THE LICENSING PROCESS This project, dealing with the migration and licensing for pathogenicity of Th lymphocytes, has been extended in FY 2016 to address comments of reviewers (J. Immunol) on our manuscript (Shedding new light on the process of licensing for pathogenicity by Th lymphocytes). In the experimental system used in these experiments, lymphocytes transgenically expressing TCR against hen egg lysozyme (HEL) are adoptively transferred into recipients in which HEL is transgenically expressed in the eyes. The transferred cells initiate inflammation in the recipient eye only when they are activated and in the experiments carried out in FY 2016, we examined systems in which the transferred cells were activated (i) in vitro, by exposure to the antigen (HEL), or (ii) in vivo, by exposure to HEL and TLR ligands. The transferred cells of the two systems require 2-3 days of licensing in organs other than the target organ (the eye), during which they proliferate AND acquire the capacity to initiate the ocular inflammation. New information was collected on three issues: (1) Donor cells activated in vitro and injected by the intraperitoneal (i.p.) route migrate with similar kinetics to the lung and the parathymic lymph nodes). Importantly, following the licensing process, the total number of donor cells in the parathymic lymph nodes was approximately x10 higher than that in the lung. Furthermore, the total number of donor cells in the spleen in these recipient mice was approximately x100 larger than that in the lung. These observations thus provide additional new data to indicate that the lung is not the organ where licensing takes place, as claimed in a recent Nature paper (Odoardi et al., 2012). (2) We also investigated the homing of donor cells to the recipients liver. The liver is the organ where dead and dying cells are eliminated, but yet, we found that small numbers of the live transferred donor cells home to the liver and undergo in this organ the licensing process, before migrating back into the circulation. This observation further supports our notion, that the licensing process takes place in multiple organs, rather than only in the lung, as claimed by Odoardi et al., 2012. (3) We used the microarray technology to compare between donor cells that acquire pathogenicity following activation in vitro and those that are activated in vivo, in their patterns of transcript expression of major inflammation-related genes prior to their invasion of the eyes. Similarities were found between the two cell populations in the patterns of expression changes by the majority of the tested genes. Since the system in which donor cells are activated in vivo could be considered a model for the physiological process of initiation of pathogenic autoimmunity, our data thus provide new information to support the notion concerning the licensing phenomenon in this physiological process. NEW INFORMATION CONCERNING THE IMMUNOSUPPRESSIVE AGENT TMP778 New information collected in FY 2016 include: (i) Treatment with TMP778 inhibits the development of EAU and its effect is attributed to the selective inhibition of the Th17 lymphocyte subpopulation. Yet, we found in new experiments in FY 2016 that treatment with TMP778 also reduces the production of interferon (IFN)-, the signature cytokine for Th1 lymphocytes. This unexpected finding of reduced levels of IFN- in lymphocytes from TMP778-treated mice was demonstrated by both the reduced levels of IFN- released by cells from treated mice, as well as by flow cytometry, showing that the proportion of spleen cells expressing intracellular IFN- was smaller among spleen cells from mice treated with TMP778 than those from the control (vehicle-treated) mice. (ii) The effect of treatment with TMP778 on the immune system was further examined by analyzing the expression of genes known to regulate the immune response by spleen cells from mice treated with TMP778 and their controls, treated with the vehicle. So far, we found that treatment with TMP778 reduces in spleen cells the expression of RORt, Tbet, RUNX3, STAT4 and Eomes, but moderately enhances the expression of IL-10. (iii) The possible involvement of increased proportion of T-regulatory (Treg) cells in the reduced immune responses in TMP778-treated mice was examined by flow cytometry and ruled out: the proportion of these cells, identified by their expression of the transcription factor FoxP3, was the same in mice treated with TMP778 and their controls, treated with the vehicle (15% of total Th cells). INHIBITORY EFFECTS OF TOFACITINIB IN VIVO AND IN VITRO Another compound with immunosuppressive capacity we examined during FY 2016 is Tofacitinib. This synthetic molecule is an inhibitor of JAK-STAT signaling pathways and has been found to efficiently inhibit the pathogenic processes of rheumatoid arthritis and similar diseases. We tested the effects of Tofacitinib on development of EAU and related immune responses in mice. The major observations include: (i) Treatment with Tofacitinib inhibited moderately the development of EAU, analyzed by histological analysis; (ii) Lymphocytes from mice treated with Tofacitinib were inferior to their controls (mice treated with the vehicle) in their production of IFN-, but not of IL-17. The differences were demonstrated by comparing both the release of the cytokines into culture supernatants and by the proportions of lymphocytes expressing the cytokines intracellularly, determined by flow cytometry. (iii) The selective inhibitory effects of Tofacitinib on generation of Th1 and Th17 was also observed in an in vitro system in which nave CD4 cells were activated by antibodies against CD3 and CD28. Adding Tofacitinib to these cultures selectively inhibited the production of IFN-, but not of IL-17. EFFECTS OF TRISTETRAPROLIN (TTP) ON THE DEVELOPMENT OF EAU AND RELATED IMMUNE RESPONSES A recent publication in PNAS (113:1865, 2016) reported that mice in which the TTP mRNA is stabilized (KI mice) are resistant to experimental autoimmune diseases. The KI mice and their wild type (WT) controls were provided to us by Perry Blackshear, NIEHS, Research Triangle Park, NC (who generated the KI mice), to be tested for their capacity to develop EAU and related immune responses. The results were remarkably unequivocal: all the WT mice developed EAU, while essentially no disease developed in the KI mice. Likewise, both the cellular and humoral immune responses of the KI mice were strikingly lower than those of the WT mice. Thus, our data shed new light on the extent of inhibition of the autoimmune response in the KI mice and support the notion that TTP could be used as an immunosuppressive agent. This study also yielded convincing data showing the higher susceptibility of female mice to autoimmunity as compared to male mice. Thus, whereas the mean level of EAU severity of the female mice (determined by histological analysis) was 2.5 +/- 0.31, that of the males was 0.95 +/- 0.21 (p =0.006). In addition, the differences between cellular immune responses of PARENT and their WT controls were remarkably more apparent between the female mouse groups than between the male groups. Further, this study provides an additional piece of interesting information: the WT (C57Bl/6) mice from the animal facility in Research Triangle Park developed EAU more consistently and with levels clearly higher than those we are routinely observing with the C57Bl/6 mice provided by Jackson Lab. A likely explanation is that the microbiome of the mice from the facility in Research Triangle Park is different from that of the mice from Jackson Lab and it better promotes the pathogenic process of EAU.
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