Steroid receptor coactivator 3 (SRC-3/AIB1) is enriched and functional in mouse and human Tregs

Abstract

A subset of CD4 + lymphocytes, regulatory T cells (Tregs), are necessary for central tolerance and function as suppressors of autoimmunity against self-antigens. The SRC-3 coactivator is an oncogene in multiple cancers and is capable of potentiating numerous transcription factors in a wide variety of cell types. Src-3 knockout mice display broad lymphoproliferation and hypersensitivity to systemic inflammation. Using publicly available bioinformatics data and directed cellular approaches, we show that SRC-3 also is highly enriched in Tregs in mice and humans. Human Tregs lose phenotypic characteristics when SRC-3 is depleted or pharmacologically inhibited, including failure of induction from resting T cells and loss of the ability to suppress proliferation of stimulated T cells. These data support a model for SRC-3 as a coactivator that actively participates in protection from autoimmunity and may support immune evasion of cancers by contributing to the biology of Tregs.

Figure 1

Steroid Receptor Coactivator 3 (SRC-3/NCOA3) is highly expressed in Tregs. (A) Ncoa3/Src-3 probe intensity in various tissues from BioGPS database (Gel image cropped from original blot submitted as supplemental figures) and the blue and red bars each represent expression values from two different probes from the microarray used in the BioGPS database. (B) Correlation of expression between NURSA coactivators and Foxp3 in Tregs using ImmunoNavigator. (C) Hematopoietic lineage map of Ncoa3/Src-3 expression showing highest levels in CD4⁺CD25⁺FOXP3⁺ Tregs in the spleen. (D) Fractionation of human blood shows elevated SRC-3 protein levels in CD4 + lymphocytes (Gel image cropped from original blot submitted as supplemental figures). (E) Enriched transcript levels of SRC-3 in freshly isolated Tregs in humans. (F) Imaging cytometry shows SRC-3 protein expression in CD4⁺CD25⁺FOXP3⁺ human Tregs.

Figure 2

SRC-3 knockdown or inhibition in human Tregs reduces FOXP3 and CD25 levels. (A) SRC-3 transactivates FOXP3 promoter in transiently transfection assay. (B) RNAi knockdown of SRC-3 in Tregs decreases transcript levels of FOXP3, CD25, CTLA-4, and PD-1. (C) SRC-3 knockdown in Tregs decreases transcript levels of SRC-3, FOXP3 and CD25 in multiple human donors. (D) Inhibition of SRC-3 with SI-2 (100 nM for 12 h) in Tregs decreases transcript levels of FOXP3 and CD25 in multiple human donors. (E) SRC-3 inhibition reduces protein expression of FOXP3 and CD25 in Tregs from multiple human donors measured using traditional flow cytometry and (F) imaging cytometry.

Figure 3

Inhibition of SRC-3 abrogates Treg suppression function. (A) Low-dose inhibition of SRC-3 with SI-2 promotes the proliferation of PHA and CD3/CD28 stimulated human PBMC and bulk CD4 + T cells. (B) Inhibition of SRC-3 activity in human resting T cells blocks induction into iTregs and (C) restricts their ability to suppress proliferating T cells. (D) SRC-3 inhibition in Tregs directly isolated from human blood donors relieves their suppressive potential.