Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε-/- mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).1.

Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing.

Tissue repair processes maintain proper organ function following mechanical or infection-related damage. In addition to antibacterial properties, mucosal associated invariant T (MAIT) cells express a tissue repair transcriptomic program and promote skin wound healing when expanded. Herein, we use a human-like mouse model of full-thickness skin excision to assess the underlying mechanisms of MAIT cell tissue repair function. Single-cell RNA sequencing analysis suggested that skin MAIT cells already express a repair program at steady state. Following skin excision, MAIT cells promoted keratinocyte proliferation, thereby accelerating healing. Using skin grafts, parabiosis, and adoptive transfer experiments, we show that MAIT cells migrated into the wound in a T cell receptor (TCR)-independent but CXCR6 chemokine receptor-dependent manner. Amphiregulin secreted by MAIT cells following excision promoted wound healing. Expression of the repair function was probably independent of sustained TCR stimulation. Overall, our study provides mechanistic insights into MAIT cell wound healing function in the skin.

Human MAIT cells are devoid of alloreactive potential: prompting their use as universal cells for adoptive immune therapy.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are semi-invariant T cells that recognize microbial antigens presented by the highly conserved MR1 molecule. MAIT cells are predominantly localized in the liver and barrier tissues and are potent effectors of antimicrobial defense. MAIT cells are very few at birth and accumulate gradually over a period of about 6 years during the infancy. The cytotoxic potential of MAIT cells, as well as their newly described regulatory and tissue repair functions, open the possibility of exploiting their properties in adoptive therapy. A prerequisite for their use as 'universal' cells would be a lack of alloreactive potential, which remains to be demonstrated. METHODS: We used ex vivo, in vitro and in vivo models to determine if human MAIT cells contribute to allogeneic responses. RESULTS: We show that recovery of MAIT cells after allogeneic hematopoietic stem cell transplantation recapitulates their slow physiological expansion in early childhood, independent of recovery of non-MAIT T cells. In vitro, signals provided by allogeneic cells and cytokines do not induce sustained MAIT cell proliferation. In vivo, human MAIT cells do not expand nor accumulate in tissues in a model of T-cell-mediated xenogeneic graft-versus-host disease in immunodeficient mice. CONCLUSIONS: Altogether, these results provide evidence that MAIT cells are devoid of alloreactive potential and pave the way for harnessing their translational potential in universal adoptive therapy overcoming barriers of HLA disparity. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number NCT02403089.

Datasets and analyses of molecular dynamics simulations of covalent binary and ternary complexes of MHC class I-related molecule/T-cell receptor (MR1/TCR) agonists to understand complex formation and conditions of fluorescent labelling.

Data of molecular dynamics (MD) simulations were obtained for mucosal-associated invariant T (MAIT) cell ligands complexed with MR1 or MR1/TCR. Ligands included in the simulations were natural ligands 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), 5-(2-oxopropylideneamino)-6-(D-ribitylamino)uracil (5-OP-RU), their C5' ethinylated analogs in S or R configuration, as well as the corresponding fluorophore-reacted products. All-atom models of the binary and ternary complexes were constructed using PDB entry 4NQE and docked poses [1]. Missing loops, N- and C-termini were completed by homology modelling, the loop conformations optimized, and the models energy minimized prior to setup for MD simulations. A standard pre-equilibration protocol was applied before the production phase of 120 ns simulation as NPT ensemble at 300 K and 1 atm applying an explicit solvent model with OPLS3 force field parameters. Atomic coordinates and energies were recorded every 60 ps and 12 ps, respectively. The corresponding raw data files of the MD simulations are part of this dataset. All simulations were analysed with respect to root mean square deviations (rmsd) and root mean square fluctuations (rmsf) of the coordinates of protein and ligand atoms, stability of protein secondary structure, protein-ligand contacts, ligand torsion profiles, and ligand properties. More detailed statistics of non-covalent interaction counts were also collected. Radial distribution functions (rdf) were calculated when relevant. Visualization of the trajectories permits appreciation of the molecular dynamics of both, ligands and proteins and their interactions, thereby supporting drug design of MAIT cell ligands; furthermore, additional analysis of e.g. conformational changes or interactions not reported in the primary publication [1] can be performed on the data. The raw data may also be used as starting point for extension of the simulations or more sophisticated MD techniques.

Synthesis, biological evaluation and molecular modelling of new potent clickable analogues of 5-OP-RU for their use as chemical probes for the study of MAIT cell biology.

MAIT cells are preset αß T lymphocytes that recognize a series of microbial antigens exclusively derived from the riboflavin biosynthesis pathway, which is present in most bacteria. The most active known antigen is unstable 5-(2-oxopropylideneamino)-6-(d-ribitylamino)uracil (5-OP-RU) which is stabilized when bound and presented to MAIT cells by MHC-related protein 1 (MR1). Here we describe the chemical synthesis and biological evaluation of new chemical probes for the study of MAIT cell biology. The two probes were ethinyl functionalized analogues of 5-OP-RU able to react through CuAAC also called "click chemistry". The molecules up-regulated more MR1 than 5-OP-RU and they efficiently activated iVα19 Vß8 TCR transgenic murine MAIT cells but not iVα19 TCRα transgenic MAIT cells indicating a surprisingly strong impact of the TRCß chain. Moreover, the use of these molecules as chemical probes was validated in vitro by efficient and selective binding to MR1 revealed via fluorescence microscopy. This study was also complemented by molecular modelling investigation of the probes and the binary/ternary complexes they form with MR1 and the TCR. These new probes will be crucial to delineate the dynamics of 5-OP-RU at the cellular or whole organism level and to identify the cells presenting 5-OP-RU to MAIT cells in vivo.