Angela Schincaglia

Mucosa-associated invariant T (MAIT) cells have been paradoxically implicated in both tissue repair and fibrosis. However, when and how they modulate fibrogenesis in the injured liver remain unclear. Here, using the carbon tetrachloride-induced model of liver injury in MR1- and MAIT cell-sufficient and -deficient mice, we identify MAIT cells as an early driver of fibrogenesis. The presence of MAIT cells exacerbated hepatocellular injury, myofibroblast activation, and matrix deposition early in the course of fibrosis development, but not at later stages. This was accompanied by rapid polarization of hepatic MAIT cells toward a MAIT17 phenotype and enrichment of pro-fibrotic transcriptional programs. Concurrently, MAIT cells acquired an exhaustion-associated phenotype while still retaining their effector functions. Mechanistically, we demonstrate that MAIT cells limit hepatic regulatory T (T _reg ) cell accumulation, accompanied by reduced Ki-67 and CXCR3 levels in the latter population, suggesting their impaired proliferation and tissue recruitment. Furthermore, T _reg cell inactivation reversed MAIT cell-dependent differences in the severity of fibrosis, establishing T _reg cells as a key downstream mediator. Together, these findings identify MAIT cells as early orchestrators of fibrogenesis and reveal a novel MAIT-T _reg axis that can be considered a potential therapeutic target in the early stages of fibrotic diseases.

Background: Mucosa-associated invariant T (MAIT) cells represent a unique population of innate-like T lymphocytes capable of detecting non-peptide antigens in the context of monomorphic antigen-presenting molecules. Due to their abundance in barrier tissues, reactivity to local inflammatory cues, and cytotoxic and regulatory functions, MAIT cells are poised to shape the dynamics of various tumor microenvironments. Growing evidence suggests that MAIT cells can exert protumor and/or antitumor effects in cancers arising from or metastasizing to mucosal tissues. However, MAIT cell roles in bladder cancer (BCa) remains unclear. Methods: To begin to identify MAIT cells in BCa, we stained bladder tumor biopsies for T cell receptor (TCR) Vα7.2 ⁺ cells. We then refined a human MAIT cell signature, which enabled us to interrogate a bulk RNA sequencing dataset and conduct correlation analyses linking intratumoral MAIT cell abundance and mortality from BCa. To extend our work to an in vivo setting, we employed a clinically relevant mouse model in which Mr1 ^+/+ B6-MAIT ^CAST (MAIT-sufficient) and Mr1 ^−/− B6-MAIT ^CAST (MAIT-deficient) mice were exposed to N-butyl-N-(4-hydroxybutyl)nitrosamine, a chemical carcinogen associated with tobacco smoke. In additional experiments, MAIT cells were functionally removed through acetyl-6-formylpterin (Ac-6-FP) administration. Effector and regulatory cell types were phenotyped by flow cytometry, and BCa tumor burden and progression were assessed by MRI and/or H&E and Ki67 staining. Results: TCR Vα7.2 ⁺ cells were readily detectable in several BCa biopsies, and our bioinformatic analyses correlated heavier MAIT cell presence in BCa tumors with poorer overall survival. Similarly, we found higher tumor burdens in Mr1 ^+/+ B6-MAIT ^CAST mice than in Mr1 ^−/− or Ac-6-FP-treated animals. Bladder MAIT cells from tumor-bearing mice exhibited phenotypic MAIT17 bias based on transcription factors they harbored along with increased interleukin-17A and tumor necrosis factor-α production capacities upon stimulation. Finally, FoxP3 ⁺ regulatory T (T _reg ) cell frequencies were elevated in Mr1 ^+/+ mouse bladder tumors, likely contributing to an immunosuppressive tumor microenvironment, a finding that could be recapitulated in our transcriptomic studies on human BCa. Conclusions: MAIT cells are abundant in BCa tumor microenvironments where they potentiate T _reg cell accumulation and play protumor roles.

Cytokinins (CKs) are small signaling molecules present in all kingdoms of life; however, their biological relevance outside of plants is poorly characterized. CK detection across kingdoms has been shown to be inconsistent and is often organism or cell line dependent. To ensure optimal detection in epithelioma papulosum cyprini (EPC), a cold-blooded vertebrate cell line, a range of biomasses were evaluated. CKs were extracted by solid-phase extraction and then detected using high-performance liquid chromatography and tandem mass spectrometry. The objectives were to establish endogenous CK profiles and characterize their biosynthesis in EPC cells. Among the 14 CKs that were scanned for, only 4 forms were detected overall within the samples analyzed, including, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), N6-(Δ2-isopentenyl) adenine-9-riboside-5′ (mono/di/tri)phosphate (iPNT), and cis-zeatin-9-riboside-5′ (mono/di/tri)phosphate ( cZNT) and 2‐methylthio‐zeatin (2MeSZ). The total CK concentrations ranged from 0.03 pmol/g to 681.025 pmol/gfwt. The results from this study provided evidence that EPC cells can produce and process CKs.

Cytokinins (CKs) are a class of growth‐promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA‐IPTs). In this study, the social amoeba, Dictyostelium discoideum , was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate‐IPT gene, iptA . The life cycle of D. discoideum is unique and possesses both single‐ and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum . To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA ‐deficiency results in cytokinesis defects, and both iptA ‐deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.