IL-10 expression by CD4+ T cells is an important mechanism to control immune responses. We previously showed that anti-TNF therapy increases the frequency of human IL-10+ CD4+ T cells in vivo and in vitro and identified IKZF3 as a putative transcriptional regulator. Here, we examined the expression of IL-10 and IKZF3 through flow cytometry and quantitative-PCR and manipulated IKZF3 expression using lentiviral overexpression and pharmacological inhibition.
IL-10 expression was increased in CD4+ T cells upon stimulation and was maintained at higher levels (mRNA and protein) upon culture with anti-TNF after 3 days. IKZF3 was expressed at higher levels in IL-10+ CD4+ T cells compared to other cytokine-producing cells both ex vivo and after CD3/CD28 activation. Pharmacological inhibition of IKZF3 using the drug lenalidomide significantly reduced the frequencies of cells expressing IL-10 after CD3/CD28 activation but was unable to affect levels of IL-10 expression ex vivo. Lentiviral over-expression of IKZF3 was not sufficient to induce IL-10 mRNA or protein expression. Furthermore, luciferase reporter assays using putative regulatory regions of the IL10 locus indicated that, unlike cMAF, IKZF3 was unable to drive reporter gene expression in isolation. Finally, we show that CD4+ T cells cultured with anti-CD3/CD28 in the presence or absence of the TNF inhibitor adalimumab have increased IL-10 expression, but no increase in the expression of IKZF3.
Our findings indicate that whilst there is an association between IKZF3 and IL-10 expression in CD4+ T cells, IKZF3 is insufficient to drive IL-10 expression.
Funded by Versus Arthritis (#21139)
Michael Ridley– Research Assoicate, Kings College London
Veerle Fleskens– King's College London
Ceri Roberts– NHS
Sylvine Lalnunhlimi– King's College London
Giovanni Povoleri– Post-Doctoral Research Associate, King's College London
Paul Lavender– King's College London
Leonie Taams– King's College London