RORγt-Mediated TNF-α Production from Human FoxP3+ Regulatory T Cells Contributes to Tissue Inflammation in Acute Viral Infection
Authors: Choi Yoon Seok , Moon Ji Young , Baek Seung Woo , Ryu Hyewon , Song Ik Chan , Lee Hyo Jin , Yun Hwan-Jung , Jo Deog-Yeon , Shin Eui-Cheol 
Published: November 2019

Abstract
CD4+CD25+Foxp3+regulatory T (Treg) cells control immune responses, thereby preventing excessive inflammation and homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. However, such inflammatory changes have not been reported so far in human acute viral infection. Herein, we investigated the production of inflammatory cytokines from Treg cells of acute hepatitis A (AHA) patients. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory conversion of Treg cells.
First, we examined the production of a variety of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4+CD25+Foxp3+Treg cells of AHA patients produced TNF-α upon TCR stimulation, particularly at the acute phase. TNF-α production by Treg cells was dramatically diminished at convalescent phase of AHA.
Next, we investigated the expression of Th cell type-specific transcription factors and chemokine receptors in TNF-α-producing Treg cells of AHA patients, using multicolor flow cytometry. TNF-α-producing Treg cells expressed substantially higher level of RORγt and CCR6 than the TNF-α-counterpart, suggesting that they share various immunophenotypes of helper 17 T (Th17) cells. More strikingly, the TNF-α production was significantly reduced by RORγt inhibition, indicating that TNF-α production from Treg cells of AHA patients is controlled by Th17-specific transcription factor RORγt.
We further examined the suppressive activity of TNF-α-producing CD4+CD25+Foxp3+ Treg cells of AHA patients. TNF-α+CD4+CD25+Foxp3+ Treg cells of AHA patients expressed lower level of Foxp3 than the TNF-α- counterpart. Similarly, the percentage of CD39+ cells was lower in TNF-α+CD4+CD25+Foxp3+ Treg cells than in the TNF-α- counterpart. In fact, Treg suppressive activity of AHA patients was significantly reduced compared with that of healthy controls when non-Treg CD4+ T cells or CD8+T cells were used as responder cells.
Finally, we examined the clinical significance of TNF-α-producing CD4+CD25+Foxp3+ Treg cells in AHA patients. In particular, we focused on the liver injury, which is mediated by effector T cells during AHA. Intriguingly, the frequency of TNF-α+ cells among circulating Treg cells significantly correlated with the serum ALT level whereas the frequency of IFN-γ+ or IL-17A+cells did not. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to severe immune-mediated liver injury in AHA patients.
Taken together, Treg cells undergo functional alteration to produce TNF-α during AHA. TNF-α-producing CD4+CD25+Foxp3+ Treg cells of AHA patients exhibit a Th17-like feature and produce TNF-α in a RORγt-dependent manner. TNF-α-producing Treg cells show reduced suppressive activity and are associated with severe liver injury in AHA. Importantly, we provide new insight into immunopathologic mechanisms in human acute viral infection, by demonstrating inflammatory conversion of Treg cells and its association with immune-mediated liver injury in AHA.

Disclosures
No relevant conflicts of interest to declare.