Introduction

Steroid receptor coactivator 3 (SRC-3) is highly expressed in T regulatory cells (Tregs). Our lab has reported that specific knock out (KO) of the SRC-3 gene in Tregs using a Foxp3^{eGFP-Cre-ERT2}/SRC-3^flox/flox mice enabled the mouse immune system to eliminate an aggressive E0771 syngeneic mouse triple negative breast cancer and prevent tumor re-emergence. The same durable anti-tumor effect was seen when SRC-3 KO Tregs were adoptively transferred to a wild type tumor bearing mouse. This evidence points to SRC-3 KO Tregs potentially having therapeutic avenue as a cancer therapeutic. Due to issues with the limited numbers of natural Tregs (nTregs) which comprise only ~5% of CD4+ T cells in the body, an attractive possibility is to induce CD4+ T conventional cells (Tconv) into Tregs in vitro. 

Methods

CD4+ CD25- T conventional (Tconv) cells were isolated from mouse spleens and induced into Tregs (iTreg) by adding TGF-beta, anti-IL-4, and anti-IFNγ in the culture medium. Western blots, immunofluorescence staining, and flow cytometry were used to characterize iTreg cells. In addition, SRC-3 KO or WT iTregs were collected and adoptively injected into tumor bearing mice to investigate the anti-tumor function of these cells. 

Results

Eighty percent of the Tconv cells were successfully converted into iTregs after four days incubation with TGF-beta, anti-IL-4, and anti-IFNγ. Similar to nTregs, iTregs also showed higher SRC-3 expression compare to their parental Tconv cells. In addition, iTregs show higher levels of BCL-2 and phosphorylated STAT-5 than nTregs, indicting their higher viability and proliferation. When adoptively injected, SRC-3 KO iTregs were able to eliminate tumors, just as SRC-3 KO nTregs did. Further study revealed that in tumors from mice who received SRC-3 KO iTregs, there was a significant increase in CD8+ cells and Granzyme B expression. 

Conclusion

SRC-3 KO iTregs are similar in their properties and their effectiveness against tumor growth when compared to SRC-3 KO nTregs. The ability to produce a large number iTregs makes them an attractive approach to study the anti-tumor effect of these modified Treg cells and makes them a potentially effective means to produce an abundance of 

SRC-3 KO Tregs for therapeutic use.