Translatome analyses by bio-orthogonal non-canonical amino acid labeling reveal that MR1-activated MAIT cells induce an M1 phenotype and antiviral programming in antigen-presenting monocytes

Abstract

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

Cellular and Biophysical Applications of Genetic Code Expansion

“…Integrating such methodologies with transcriptomic analysis could further clarify the immune response mechanisms to infectious diseases. 545…”

“…This investigation deepened the understanding of protein expression alterations in immune cells induced by MR1 ligands, including their antiviral responses. Integrating such methodologies with transcriptomic analysis could further clarify the immune response mechanisms to infectious diseases …”

Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation

“…The studies highlighted above demonstrate BONCAT’s potential in therapeutic research for understanding parasite behaviors and responses to potential treatments . Tangentially, BONCAT has also been used to monitor the responses of mammalian cells to injury and treatments. − Monteiro and colleagues have used it to analyze the resistance of lung cancer cells to the drug cisplatin and determine the potential targets to monitor in epigenetic resistance in lung cancer . Shah et al have also used it to measure the protein synthesis and transport dynamics in the retina before and after optic nerve injury.…”