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M. Aziz, C. Tan, B. P. Vistica, G. Shi, J. D. Lovaas, I. Gery; Generation, Unique Features and Pathogenicity of T-Helper Cells Selectively Producing Il-9 ("th9"). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2017.
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Recently, lines of Th cells selectively expressing IL-9 were generated in vitro and were designated "Th9". These line cells originated from wild type donors and were non-antigen-specific. Here, we generated antigen-specific "Th9" lines and analyzed their pattern of cytokine response and capacity to induce inflammation in eyes expressing the target antigen.
Naive CD4+ cells isolated from hen egg lysozyme (HEL)-specific TCR transgenic mice were activated by HEL and polarized toward IL-9 production by IL-4/TGF-β. Real-time PCR was used to measure mRNA transcript levels, whereas ELISA and intracellular staining were used to measure cytokine production. Pathogenicity of Th9 cells was assessed by their capacity to induce ocular inflammation in recipients expressing HEL in their eyes. Injected Th9 cells were also tested for their proliferation in recipient spleens.
Th9 lines produced high levels of both IL-9 and IL-10, but with striking differences in their kinetics. Whereas IL-9 mRNA transcript reached its peak on day 2 of activation and declined sharply after day 3, IL-10 transcript reached its peak on day 3 and retained that level throughout the 6-day testing period. Moreover, intracellular staining of IL-9 reached its peak, of ~45% positive cells, on day 3 and dropped to less than 10% on day 4, whereas intracellular IL-10 increased gradually, reaching a peak of ~40% on day 6. When adoptively transferred to recipients expressing HEL in their eyes, HEL-specific Th9 cells induced ocular inflammation. Importantly, a good correlation was observed between IL-9 production kinetics and the pathogenic capacity of Th9 cells. Th9 cells collected only on day 3 produced inflammation in recipient eyes, whereas no disease was detected in recipients of Th9 cells collected on day 4. Th9 cells harvested on day 3 of culture proliferated vigorously in the recipient spleen before invading the target eye. In contrast, Th9 harvested on day 4 failed to demonstrate a similar proliferative response.
Antigen-specific Th9 lines demonstrate a unique pattern of cytokine production and induce ocular inflammation only when collected at their peak of IL-9 production.
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