Control of regulatory T cell activity by endogenous and pathogen-derived EGFR ligand

Abstract

Regulatory T cells (Treg) are CD4+ FOXP3+ T lymphocytes whose function is to prevent autoimmunity and immune cell-mediated damage on infected tissues. They carry out their suppressive effects either by direct cell-cell contact or through the use of soluble mediators such as IL-10 and TGFb. Activated Treg express the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor found in most tissue types and leukocytes. EGFR signalling induces behavioural changes in Treg: in particular, the ligand Amphiregulin (AREG) is required for optimal Treg function in various in- vitro and in-vivo experimental models. The aim of my thesis has been to explore the effects of EGFR signalling on Treg function in the context of infection, induced either from endogenously expressed or pathogen-derived ligands.

In Chapter 2 I examined the effects of Vaccinia (VACV) virus-derived Vaccinia Growth Factor (VGF), a pathogen-derived soluble protein with EGF-like activity, on Treg function. I showed that Treg function is increased when exposed to UV- inactivated Vaccinia virus, but that this activation can be blocked by deleting EGFR expression on Treg. Using an in-vivo pneumonitis model, I showed that mice deficient in EGFR expression on their Tregs were more resistant to VACV infection compared to wildtype, and that following infection there was less bioactive TGFb in the bronchoalveolar lavage supernatant of these animals. Taken together, these data imply that Vaccinia virus derived VGF enhances the suppressive capacity of Tregs and use this enhanced activation as a means of immune escape.

In Chapter 3 I used the filariasis model Litomosoides sigmodontis (a chronic helminth infection of rodents) to examine the effects of EGFR signalling in Tregs induced by the endogenous ligand Amphiregulin, and to study the effects of EGFR blockade on the helminth-specific immune response using the commercially available tyrosine kinase inhibitor Gefitinib. We found that EGFR deletion on Treg lead to no increase in TH2 cytokine production or parasite rejection. Also, complete deletion of Amphiregulin in Areg-/- mice had no significant effect; nor did the administration of Gefitinib over several weeks in WT mice. From these findings, we conclude that AREG-EGFR signalling on Tregs (or EGFR signalling in general) is not a significant factor influencing the immune response to L.sigmodontis infection.

In Chapter 4 I examined the effects of the complete deletion of Amphiregulin in Areg-/- mice, and the deletion of Amphiregulin in specific cell types, on the activity of activated Tregs in an airway allergy model. To induce and activate Tregs we used the murine helminth Heligmosomoides polygyrus, which infects the gastrointestinal tract but also induces Treg expansion systemically. Previous work in our group had determined that Amphiregulin deletion in mice led to a decreased Treg-mediated immunosuppression following H. polygyrus infection and airway OVA challenge, as evidenced by greater eosinophilia in the lungs of Areg-/- than wildtype mice. However, I failed to replicate the initial findings of our group’s work, seeing no difference in Treg activity or TH2 response between infected and noninfected animals, and also no significant differences in different mouse strains with a cell type-specific deletion of Amphiregulin.

Taken together, my thesis provides evidence for the enhanced suppressive capacity of Tregs by a viral pathogen derived EGFR ligand, in order to create a local immunosuppressive environment. To our knowledge, this is the very first time such an immune escape mechanism has been demonstrated. It also demonstrates that in the face of helminth-mediated activation of Treg, EGFR signalling is not a significant factor contributing to local immune suppression. This further clarifies the role of EGFR signalling on Tregs in the setting of both acute and chronic infection.