Methods: Naïve CD4⁺ cells were isolated from wild-type (WT) and T-bet null (no IFN-Y expression) mice, and then adoptively transferred into Rag1 ^-/- mice, respectively. The Rag1 ^-/- recipients were subsequently exposed to desiccating stress for 14 days followed by housed in the standard vivarium for additional 3 weeks. At the end of the follow up, the bulbar conjunctiva and DLNs of Rag1 ^-/- mice were collected, and mTh17 frequencies were examined by flow cytometry. Furthermore, eTh17 and eTh17/1 cells were isolated from DLNs of WT DED mice and cultured in medium supplemented with IL-23 for 48 hours, and the frequencies of mTh17 cells were assessed by flow cytometry. To evaluate the in vivo blockade effect, IL-23 signaling was blocked by intraperitoneal injection of anti-IL-23R antibody at day 14 when acute DED mice were returned to the normal environment. The disease severity was then evaluated for 7 days by corneal fluorescein staining, and mTh17 cells were examined using flow cytometry.

Results: Rag1 ^-/- mice receiving naïve CD4⁺ cells isolated from T-bet null mice did not generate eTh17/1 cells by day 14, and developed significantly lower frequencies of mTh17 in both DLNs and conjunctiva by day 35 (chronic DED), compared to those receiving WT naïve CD4⁺ cells (p<0.05). In vitro culture of eTh17/1 in the presence of IL-23 led to a prominent generation of mTh17 as 2 times high as eTh17 culture. Finally, in vivo blockade of IL-23 signaling prevented the development of mTh17 cells from existing effector Th17 cells, and significantly decreased ongoing disease severity by 33% (p = 0.004).

Conclusion: Our data suggest that eTh17/1 is the predominant effector subset converting to mTh17 mediated by IL-23, and blockade of IL-23 could be a promising therapeutic strategy for chronic DED.

Disclosure: N. This study was supported by grant: NIH R01 EY20889.

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