Interferon Regulatory Factor 8 (IRF8) antagonizes IL-10 and CTLA4 production in T cells

Abstract

Purpose : Interferon regulatory factor 8 (IRF8) and IRF4 are two members of the IRF family of transcription factors that are recruited to AP1-IRF composite elements (AICEs) through interactions with Basic leucine zipper transcription factor, ATF-like (BATF). In T cells, recruitment of IRF4 to AICE elements in il10 or ctla4 locus promotes transcription of il10 or ctla4. In this study, we investigated whether IRF8 also regulates IL-10 and CTLA4 expression in T cells.

Methods : CD4⁺ T cells were isolated from the C57BL/6 or CD4^creIRF8^fl/fl mice. The cells were activated with CD3/CD28 antibodies and characterized by FACS, intracellular cytokine staining assay and ELISA. The interaction between IRF8 and BATF was examined by Western blotting and immunoprecipitation (IP) assays. Recruitment of IRF8 to AICEs within il10 or ctla4 promoter was assessed by Chromatin IP (ChIP) assay and EMSA (electrophoretic mobility shift assay).

Results : We show here that the level of IRF8 in resting T cells is very low but is rapidly upregulated in response to cell activation. Results of our ChIP analysis suggest that IRF8 is recruited to the AICE motifs on the il10 and ctla4 promoter. Compared to the WT mice, we observed significant up-regulation of IL-10 and CTLA4 production by CD4^creIRF8^fl/fl mice. This result was confirmed at the RNA level by RT-PCR/qPCR and at the protein level by intracellular cytokine staining assay and ELISA. Interestingly, increase in the expression of these immune suppressive proteins in response to TCR activation was rapid and declined precipitously 72 hours after TCR stimulation. It is however of note that the loss of IRF8 did not interfere or antagonize interaction between IRF4 and BATF.

Conclusions : We show for the first time that IRF8 regulates the expression of IL-10 and CTLA4, two immune suppressive proteins that inhibit the development of autoimmune diseases by preventing unbridled activation of T cells. The peak increase in IL-10 and CTLA4 expression occurred 48 h after activation of IRF8-deficient T cell and diminished thereafter, suggesting that IRF8 might promote T cell activation induced by TCR/CD28 signaling while antagonizing suppressive effects mediated by CTLA4 and/or IL-10 at the early stages of activation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.