Dottorato in SCIENZE DELLA VITA

Titolo della tesi: THE ROLE OF TUMOR NECROSIS FACTOR α IN REGULATORY T CELL EXPANSION

Tumor necrosis factor (TNF) is a pleiotropic cytokine well known for its pro inflammatory effect, though several studies have highlighted its anti-inflammatory and immunomodulatory effects. It has been described that TNF promotes survival, proliferation, and effector function of regulatory T cells (Tregs), a CD4 T cell subset with suppressive function. The aim of this project is to dissect the role of TNF production by Tregs on their own expansion and function, especially in tumor, where Tregs are potentially more responsive to this cytokine because in tumor microenvironment they express higher levels of the TNF receptor TNFR2. First, we demonstrated that both mouse and human Tregs produced TNF when briefly restimulated ex vivo. We observed that Treg-derived TNF plays a role in Tregs proliferation: indeed, an anti-TNF neutralizing Ab suppressed Tregs proliferation and TNFR2 expression, suggesting the existence of a positive feedback mechanism. In murine tumors, we found that TNFR2 and TNF were both upregulated by Tregs especially at the tumor site; however, TNF and TNFR2 showed a mutually exclusive expression, suggesting the existence of two alternative states or subsets of Tregs that either sense or produce TNF. A kinetics experiment revealed that TNFR2- Tregs produced more intracellular TNF protein as early as 1.5 hours, and up to 4 hours from stimulation. Furthermore, TNF protein could be detected in the supernatant of TNFR2- Tregs two hours after PMA/ionomycin stimulation. After in vitro activation, TNFR2- Tregs maintained their capacity to produce TNF at significantly higher levels than TNFR2+ Tregs, despite upregulating TNFR2. To investigate the mechanisms behind the dichotomy between TNFR2+ and TNFR2- Tregs, we hypothesized a post transcriptional control of TNF expression in the two Tregs subtypes, since sorted TNFR2+ and TNFR2- Tregs differed for the TNF protein levels but had similar TNF mRNA at 4 hours post-restimulation. Interestingly, TNFR2+ Tregs expressed higher levels of miR-146a, which plays key roles in Treg functions and is known to repress TNF synthesis. MiR-146a-5P antagomir partially rescued TNF protein production in TNFR2+ Tregs, suggesting its role for TNF regulation in this Treg subset. If we cocultured TNFR2+ and TNFR2- Tregs to put them directly in competition, we could observe that the two populations maintained their diversity, even if TNFR2+ Tregs greatly outnumbered the TNFR2- ones, indicating a competitive advantage of the former. Furthermore, TNFR2+ Tregs transmitted their cellular material to TNFR2- Tregs and this resulted into a change in their phenotype and, perhaps, a more activated attitude. Finally, a gene expression analysis of the two Treg subsets revealed that TNFR2+ Tregs had a stronger profile of proliferation, glycolytic metabolism, and regulatory identity, compared to TNFR2- Tregs, making TNFR2+ Tregs the Treg population with a molecular program that supports proliferation and survival features. Overall, our data indicate that Tregs may exist in two functional states, characterized by different regulatory identity, and that internal regulatory mechanisms may dictate the balance between the two.