Discovery of novel MR1-dependent MAIT cell responses in infections.

Mucosal-associated invariant T (MAIT) cells are multifunctional effector cells recognizing MR1-binding metabolites. This MR1 pathway remains, despite numerous studies, incompletely understood regarding activation and induction of immune responses and was therefore further investigated in this doctoral thesis. Although the activation of MAIT cells was originally only associated with bacterial riboflavin derivatives, increasing evidence points to an MR1-dependent activation of MAIT cells in the presence of various molecules including self-antigens. Recently, the Clostridioides difficile (C. difficile)-derived toxin CDT was shown to promote MAIT cell cytotoxicity in an MR1-dependent manner, indicating a non-canonical activation of MAIT cells by CDT. In this doctoral thesis, the underlying mechanism was now specified. The substrate binding and pore-forming subunit CDTb was found sufficient to provoke MAIT cell activation in absence of the enzymatic component CDTa. Moreover, CDT stimulation of monocytes in a bicellular system with MAIT cells increased MAIT cell activation and cytotoxicity. Importantly, this activation was dependent on MR1 presentation. To further understand MR1-mediated immune responses of MAIT cells and monocytes upon antigen stimulation, a translatome study based on Bio-orthogonal non-canonical amino acid tagging (BONCAT) and ultrasensitive proteomics was established (BONCAT proteomics). MAIT cells and THP-1 monocytes were co-cultured and stimulated with the MR1-binding MAIT cell-activating metabolite 5-OP-RU as well as the inhibitory MR1 ligand Ac-6-FP. This bicellular stimulation allowed for the first time the comprehensive investigation of antigen-induced MAIT cell responses at the protein level and the first omics characterization of MAIT-interacting cells. Over 2,000 MAIT and 3,000 THP-1 active protein translations were identified by mass spectrometry following MR1 stimulation. Conjugation frequencies as well as translation were strongly increased only upon 5-OP-RU but not Ac-6-FP stimulation. Ac-6-FP regulated the protein translations of GSK3B, ADAM10, or MIF in MAIT cells, which indicated the induction of an anergic phenotype. In contrast, 5-OP-RU promoted a pro-inflammatory translatome profile in MAIT and THP-1 cells. Next to known effector responses, a distinct type I and type II Interferon-driven translatome profile was uncovered in both cell types following 5-OP-RU stimulation. Consequently, the translatome data indicated that MR1-activated MAIT cells facilitate an M1 polarization of monocytes. This phenotype was confirmed by gene and surface expression profiles of CXCL10, IL-1, CD80, and CD206. Most interestingly, the M1 polarization and Interferon-driven translatome were accompanied by the induction of an antiviral phenotype in THP-1 cells, which suppressed viral replication upon conjugation with MR1-activated MAIT cells. Thus, translatomics revealed that MR1-activated MAIT cells do not only possess anti-bacterial but also anti-viral functions and directly influence the phenotype of monocytes. In the further course of the doctoral thesis, the same BONCAT-based approach was then used to identify known and new effector proteins secreted by MAIT cells with high selectivity. Notably, several tissue repair-associated molecules were identified to be secreted independently from MR1 stimulation, including THBS1, VTN, or PLG. This indicates a pro-regenerative function of MAIT cells in the maintenance of tissue homeostasis, changing phenotypically and functionally during infection. In the last part of this thesis, the regulation of MR1-dependent MAIT cell responses was investigated by analyzing the role of CD26, which was considered a MAIT cell marker before. CD26 co-stimulation along MR1/ T cell receptor (TCR)-dependent engagement of MAIT cells was found to promote the release of cytotoxic effector molecules, suggesting CD26 as an important mechanism to control MR1-dependent MAIT cell degranulation. CD26-dependent cytotoxicity was also observed upon co-stimulation with the MERS-CoV Spike protein, whereas MAIT cells were resistant to MERS-CoV infection. This implies that MAIT cell activity during infection may be directly modulated by CD26-targeted pathogens. Although pathogen binding to CD26 could thus directly promote the killing of infected cells, it also suggests that the cytotoxicity of MAIT cells must be tightly regulated during inflammation to prevent tissue damage. In conclusion, four novel MR1-dependent mechanisms were discovered in this thesis: i. The non-canonical MR1-dependent activation of MAIT cells following CDT stimulation is mediated by monocytes. ii. Using BONCAT proteomics MR1-activated MAIT cells were found to elevate their global protein translation and to support the induction of an M1 and antiviral phenotype in MAIT-interacting monocytes. iii. MAIT cells secrete wound healing-associated candidates especially in the absence of TCR stimulation, suggesting a novel bystander function of the cells in tissue. iv. MR1-dependent MAIT cell cytotoxicity can be modulated by the abundant co-stimulatory and MERS-CoV-interacting molecule CD26.