UC Berkeley

The Toll-like receptors (TLRs) are a family of pattern recognition receptors with a well-established role promoting activation and cytokine production by innate immune cells. Expression of TLRs on T cells is controversial, with conflicting reports of various functional outcomes of T-cell intrinsic TLR signaling. Using a series of novel TLR reporter mice I have identified TLR7, a sensor of single-stranded RNA, as the only TLR expressed at detectable levels on T cells.

This dissertation seeks to characterize the role of TLR7 signaling on T cells. Stimulation of CD8+ T cells with TLR7 ligand boosts TCR activation-induced proliferation and effector cytokine production. This result suggests an alternative means of co-stimulation that could be beneficial during immune responses to viral infections. While this finding provides needed clarity to the field, it matches previously proposed theories for the role of TLR signaling on T cells, prompting me to focus instead on the role of TLR7 on Foxp3+ regulatory T cells (Tregs).

The pattern of TLR7 expression on Tregs suggested a critical functional role. Returning to the TLR7 reporter mice, I demonstrated that TLR7 is expressed by nearly all Tregs upon exit from the thymus. Tregs in the lymphoid tissues are bimodal for TLR7 expression, while those in non-lymphoid tissues tend to downregulate TLR7. Intriguingly, expression of TLR7 on Tregs is anticorrelated with antigen experience, implying that this receptor might be most critical on naïve Tregs.

The functional impact of TLR7 signaling on Tregs was novel and unexpected. Tregs in non-lymphoid tissues are capable of promoting repair following injury or infection, a phenotype characterized by production of the EGF family member amphiregulin. The stimuli that cause Tregs to upregulate amphiregulin and accumulate in damaged tissue are not well understood. Using a series of in vitro experiments, I established that Treg-intrinsic TLR7 signaling has no impact on suppressive capacity, but instead results in proliferation and elevated production of amphiregulin. These results suggest that TLR7 signaling primes Tregs for tissue repair.

To test the role of TLR7 on Tregs in vivo, I generated mice with a Treg-specific deletion of TLR7. When challenged with influenza, an ssRNA virus capable of stimulating TLR7, these mice exhibit severe disease and impaired lung function. Single-cell RNA sequencing of Tregs from influenza experiments indicate that TLR7 is important for maintenance of a population of lung-resident Tregs with that express elevated levels of amphiregulin. This data demonstrates that TLR7 signaling can directly enhance the tissue repair capacity of Tregs, a critical aspect of the host response to respiratory infections. Additionally, the ability of TLR7 to recognize self-RNA in certain contexts led me to test its role on Tregs in non-viral lung damage models. I again observed impaired lung function in mice with the Treg-specific deletion of TLR7. This result suggests a mechanism that may enable Tregs to sense and repair tissue damage more generally.

My exciting findings about the role of TLR7 on Tregs are applicable to humans as well. The final chapter of this dissertation shows that TLR7/8 stimulation of human Tregs drives proliferation and amphiregulin production, the first known method for driving human Tregs to adopt a phenotype consistent with tissue repair. This result opens the door to a new type of cell-based Treg therapy, which may be used to treat tissue damage induced by, for example, severe viral infection.