Targeted at learning about inflammatory eye diseases, this project focused in FY 2008 on the involvement in ocular inflammation of the recently discovered subset of T-helper cells, designated """"""""Th17"""""""". Cells of this subset are identified by their selective expression of interleukin-17 (IL-17), a feature that differentiates them from interferon (IFN)-gamma producing Th1, or IL-4 producing Th2 cells. The specific issues addressed in FY 2008 included: (1) Inflammatory processes induced by Th1 or Th17 cells differ in their kinetics and (2) Th17 may switch phenotype, but Th1 do not.? ? In the experimental system we used in these studies, inflammation develops in eyes of transgenic (Tg) mice expressing hen egg lysozyme (HEL) in their eyes, following adoptive transfer of T-lymphocytes specific against HEL. These T-lymphocytes are collected from another line of Tg mice, designated """"""""3A9"""""""", in which most T-cells express a HEL-specific T-cell receptor (TCR). When activated in culture in the presence of certain """"""""cocktails"""""""", i.e., mixtures of cytokines and antibodies, naive cells acquire the phenotype of Th1 or Th17, thus allowing us to obtain lineages of Th1 or Th17 cells and investigate their biological activities.? ? 1. Analysis of the pathogenic processes induced by Th1 or Th17 cells revealed remarkable differences between the pathogenic processes triggered by the two cell lineages. These differences included: (i) Th1 cells divided in recipient mice considerably faster than Th17, as measured by CFSE dilution rate; (ii) Th1 invaded the recipient mouse eyes earlier than Th17 cells: onset of the inflammatory process was day 3 for Th1 and day 4 for Th17 cells; (iii) the number of donor cells declined and the inflammation severity receded rapidly in Th1 recipient mouse eyes, whereas the number of Th17 donor cells did not decline and the inflammatory process was sustained throughout the 15 day testing period; (iv) the decrease in Th1 cells in the inflamed eye was apparently due to their high susceptibility to restimulation-induced cell death, as demonstrated by in vitro apoptosis assays; (v) Th1- and Th17-induced inflammation differed in the profile of recruited cells, with higher proportions of neutrophils in Th17-induced disease and higher CD8 infiltration in Th1-induced inflammation. ? ? 2. Our experimental system was used to resolve an enigmatic issue, namely, the source of double positive T-cells, expressing both IFN-gamma and IL-17. Double positive cells are found at inflammation sites in both humans and experimental animals and their source has remained unknown since their discovery. We approached this issue by injecting polarized populations of HEL-specific Th1 or Th17 cells into naive recipients expressing HEL in their eyes. We then collected the inflamed eyes and analyzed the infiltrating donor cells for their cytokine production. Only IFN-gamma expressing cells were found in recipient eyes injected with Th1 cells. In contrast, substantial proportions of donor cells were found to express both IFN-gamma and IL-17, or only IFN-gamma, in eyes of Th17 recipients. ? ? We further analyzed the difference between Th1 and Th17 cells in vitro, by testing these populations for their phenotype retention following additional incubation in polarization """"""""cocktail"""""""" of the opposite lineage. Again, Th1 cells retained completely their phenotype and expressed only IFN-gamma following incubation with the Th17 polarization cocktail, while the majority of Th17 switched phenotype and expressed IFN-gamma, or both IFN-gamma and IL-17 following incubation with the Th1 cocktail. Furthermore, we found that the majority of Th17 switched phenotype even following activation in the absence of cytokines and the switching was enhanced by adding to the culture the major component of the Th1 cocktail, IL-12, but partially prevented by addition of the major components of the Th17 cocktail, IL-6 and TGF-beta.? ? The phenotype switching of Th17 cells was also indicated by the expression of T-bet, the transcription factor specific to Th1 following incubation with the Th1 cocktail. In contrast, Th1 cells did not express any ROR-(gamma)t, the transcription factor specific to Th17, following incubation with the Th17 cocktail.? ? Our data thus resolved the old enigma concerning the source of double positive Th cells: they originate only from Th17.
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