Targeted at learning about pathogenic processes of inflammatory eye diseases, this project focused in FY 2010 on populations of T-cells involved in these processes. We investigated two populations of recently identified T-helper cells, specifically producing IL-17 (""""""""Th17"""""""" cells), or IL-9 (""""""""Th9"""""""" cells). In addition, we analyzed the role of the SLAT/Def6 gene in immune-mediated ocular inflammation. 1) Th17 Cells. We extended our studies aimed at further analysis of the immunobiological features of the two subpopulations of Th17 cells. In previous studies, detailed in our FY 2008 and FY 2009 Reports we described new procedures we developed for the generation of the two subpopulations of Th17 cells, one pathogenic the other is not. The experimental system we use consists of two lines of transgenic mice. T-cells of one line, designated 3A9, express T-cell receptor (TCR) specific against hen egg lysozyme (HEL), while mice of the other line, named HEL-Tg, express HEL in their eyes. When acquiring pathogenicity, T-cells of the 3A9 line induce inflammation in eyes of the HEL-Tg recipients. The pathogenic subpopulation of Th17 cells is generated by activation of naive CD4 lymphocytes of 3A9 mice with HEL and antigen-presenting cells (APC), whereas the non-pathogenic subpopulation is generated by activation of the naive CD4 lymphocytes by antibodies against the surface molecules CD3 and CD28. Both subpopulations are polarized by incubation with the cytokines IL-6 and TGF-beta. This study was extended in FY 2010 as described below. (i) Cells of the pathogenic subpopulation produce large amounts of the cytokine IL-22. To examine the possibility that IL-22 participates in the pathogenic effects of these cells, we treated recipients of the cells with antibody against IL-22. The antibody had no effect on the pathogenic effect of the Th17 cells, thus suggesting that IL-22 plays no significant role in the pathogenic capacity. (ii) Immunostaining of the pathogenic Th17 cell line revealed a clear separation between cells that produce IL-22 and those that produce IL-17. This observation thus suggests the existence of a subset within the subpopulation of pathogenic """"""""Th17"""""""", that selectively produces IL-22. It is of note that T-helper cell lines specifically producing IL-22 were described with human cells, but not with mouse cells and our study thus identifies for the first time IL-22 producing mouse T-cells. (iii) Published studies indicate that IL-23 is critical for the generation of pathogenic Th17. To investigate the participation of IL-23 in the generation of pathogenic Th17 in our system we added this cytokine to cultures in which non-pathogenic Th17 were generated (by activation with plate-bound antibodies). No pathogenicity was generated in these cultures. In contrast, addition of APC and HEL to these cultures did generate pathogenicity. We further showed that the activity of APC/HEL is apparently mediated by cell-cell activation, rather than release of IL-23, since the induction of pathogenicity was not affected by addition of anti-IL-23 antibody to the culture medium. (iv) The non-pathogenic subpopulation of Th17 exhibited a moderate level of immunoregulatory activity, shown by their capacity to inhibit the pathogenic effect of the pathogenic Th17 cells. 2. Th9 cells. Our FY 2009 Report summarized our preliminary observations concerning Th9 cells, lymphocytes that specifically produce the cytokine IL-9. Research carried out in 2010 yielded the following new information: (i) Further analysis of the Th9 pathogenicity revealed that these cells do induce ocular inflammation when adoptively transferred into recipient mice expressing the target antigen (HEL) in their eyes, but only when tested on day 3 in culture, at the peak of the IL-9 production. Importantly, no disease was induced by Th9 cells harvested just one day later (day 4), at the time point when IL-9 production was sharply reduced, as recorded in our FY 2009 Report. (ii) Analysis of the cells infiltrating the inflamed recipient mouse eyes revealed that, unlike observations with Th1- or Th17- induced ocular inflammation, no IL-9 producing cells were detected in eyes with Th9-induced inflammation. This finding is in line with the observation we made previously (recorded in FY 2009), that IL-9 production is unusually short lived following the cell activation. This assumption is also supported by our finding that Th cells expressing IL-9 were detected in the recipient blood, where no target antigen is present and no activation could take place. (iii) Th9 cells retain their phenotype, despite the reduction in IL-9 production following activation. This feature was indicated by our finding that re-stimulation of Th9 cells expressing no detectable IL-9 (following extended time in culture) responded by a typical secondary immune response, of IL-9 production at faster pace and higher levels than those of the primary response. (iv) Incubating Th9 cells in media with polarizing cytokine specific for other Th phenotypes revealed low levels of switching to Th1 or Th17 phenotypes, but considerable switching levels to the Th2 phenotype. 3. Participation of the SLAT/Def6 gene in immune-mediated ocular inflammation. The essential role of the SLAT/Def6 gene in pathogenic immune processes was demonstrated in studies with mice deficient in this gene, that were found to be inferior to wild-type controls in their capacity to develop cell-mediated immune response. We are developing a colony of the SLAT/Def6 deficient mice and preliminary data show that their capacity to develop experimental autoimmune uveitis (EAU) is significantly lower than that of their wild-type controls.

National Institute of Health (NIH)
National Eye Institute (NEI)
Investigator-Initiated Intramural Research Projects (ZIA)
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Tan, Cuiyan; Wandu, Wambui S; Lee, R Steven et al. (2017) Shedding New Light on the Process of ""Licensing"" for Pathogenicity by Th Lymphocytes. J Immunol 198:681-690
Canna, Scott W; Shi, Guangpu; Gery, Igal et al. (2017) Chronic, Systemic Interleukin-18 Does Not Promote Macular Degeneration or Choroidal Neovascularization. Invest Ophthalmol Vis Sci 58:1764-1765
Hinshaw, Samuel J H; Ogbeifun, Osato; Wandu, Wambui S et al. (2016) Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration. Invest Ophthalmol Vis Sci 57:1441-7
Richard, Arianne C; Tan, Cuiyan; Hawley, Eric T et al. (2015) The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells. J Immunol 194:3567-82
Richard, Arianne C; Tan, Cuiyan; Hawley, Eric T et al. (2015) Correction: The TNF-Family Ligand TL1A and Its Receptor DR3 Promote T Cell-Mediated Allergic Immunopathology by Enhancing Differentiation and Pathogenicity of IL-9-Producing T Cells. J Immunol 195:5839-40
Wandu, Wambui S; Tan, Cuiyan; Ogbeifun, Osato et al. (2015) Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response. PLoS One 10:e0128906
Tan, Cuiyan; Wei, Lai; Vistica, Barbara P et al. (2014) Phenotypes of Th lineages generated by the commonly used activation with anti-CD3/CD28 antibodies differ from those generated by the physiological activation with the specific antigen. Cell Mol Immunol 11:305-13
Zhao, Chan; Shi, Guangpu; Vistica, Barbara P et al. (2014) Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC. Cell Immunol 290:179-84
Nugent, Lindsey F; Shi, Guangpu; Vistica, Barbara P et al. (2013) ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity. Invest Ophthalmol Vis Sci 54:7463-9
Shi, Guangpu; Vistica, Barbara P; Nugent, Lindsey F et al. (2013) Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands. J Immunol 191:415-23

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