Control of Foxp3+ Treg Production, Stability and Function by the Nuclear Co‐regulator, Sin3A
Authors: Christensen Lanette M. , Akimova Tatiana , Han Rongxiang , Wang Liqing , Samanta Arabinda , Hancock Wayne W. 
Published: May 2022

PurposeFoxp3+ T‐regulatory (Treg) cells are well established as a crucial component of immune homeostasis and self‐tolerance. Histone/protein deacetylase (HDAC) activity within Foxp3+ Tregs plays an important role in regulation of gene expression. HDAC1 and ‐2 regulate Foxp3 expression and play critical roles in the control of Treg functions and properties. Less understood, are the contributions made by proteins which form large complexes with HDAC1 and ‐2, such as the paired amphipathic helix protein, Sin3a.MethodsWe sought to evaluate the contributions Sin3a makes to Tregs by undertaking its conditional deletion in Foxp3+ cells.ResultsSin3a‐/‐Foxp3Cre mice developed severe, systemic autoimmunity, leading to death within 2‐3 weeks of birth. These mice had greatly enlarged superficial lymph nodes, massive inflammatory lesions in lungs, skin, livers and kidneys and developed autoantibodies to islet cells, striated muscles, keratin, endomysium and gastric parietal cells. Percentages of Foxp3+ Tregs in the secondary lymphoid organs of Sin3a‐/‐Foxp3Cre mice were vastly lower than in controls (p values ranging from >0.01 to >0.0001). Further, the amount of Foxp3 mRNA expression (>20‐fold) and Foxp3 protein per Treg (>3‐fold) were also significantly reduced within Sin3a deficient animals. The ratios of T effectors to Tregs, and follicular T effectors to follicular Tregs were drastically increased within the spleens and superficial lymph nodes (ranging from 87‐154% and 152‐160%, respectively). Sin3a deficient Tregs produced significant amounts of pro‐inflammatory cytokines, including Granzyme B, IFN‐γ and IL‐2 (ranging from 12‐45% of cells). Tregs lacking Sin3a had impaired suppressive function against CD4+ and CD8+ responders (p<0.01 and p<0.05 respectively, compared to WT Tregs) in vitro. In addition, we found that Sin3a was essential for peripheral iTreg conversion since conventional CD4+CD25‐ cells from Sin3a‐/‐FoxP3Cre mice completely lacked the ability to convert to Foxp3+ iTregs ex vivo in the presence of IL‐2, TGFb, and CD3/CD28 beads. Consistent with impaired Treg function, CD4+ and CD8+ T cells from Sin3a‐/‐Foxp3Cre mice showed increased activation ex vivo (Ki67+ and CD69+ increased 120% and 230%, respectively) and enhanced production of Granzyme B and cytokines IFN‐γ and IL‐2 upon stimulation in vitro. To gain a more complete understanding of the regulatory role of Sin3a in Tregs, we are in the process of performing RNAseq. To address the vast reduction of Foxp3+ Tregs within Sin3a‐/‐FoxP3Cre mice we are investigating methylation of the CNS2 intronic region of Foxp3 by bisulphite conversion and determining the fate of Tregs in vivo by lineage tracing.ConclusionsThese data show that the nuclear co‐regulator, Sin3a, functions in modulating Treg gene expression and helps maintain the unique properties of these key immune cells. Our results indicate a potential for therapeutic modulation of Treg functions by pharmacologic targeting of components within the Sin3a complex.