Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7⁺ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

The Aryl hydrocarbon receptor (AhR) is a critical regulator of both innate and adaptive immune responses, with potent immunomodulatory effects that makes this receptor an attractive molecular target for novel therapeutics. Accumulating evidence indicates that diverse—both host’s and microbial—tryptophan metabolites profoundly regulate the immune system in the host via AhR, promoting either tolerance or immunity, largely as a function of the qualitative and quantitative nature of the metabolites being contributed by either source. Additional findings indicate that host and microbiota-derived tryptophan metabolic pathways can influence the outcome of immune responses to tumors. Here, we review recent studies on the role and modalities of AhR activation by various ligands, derived from either host-cell or microbial-cell tryptophan metabolic pathways, in the regulation of immune responses. Moreover, we highlight potential implications of those ligands and pathways in tumor immunotherapy, with particular relevance to checkpoint-blockade immune intervention strategies.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates numerous cellular responses. Originally investigated in toxicology because of its ability to bind environmental contaminants, AhR has attracted enormous attention in the field of immunology in the last 20 years. In addition, the discovery of endogenous and plant-derived ligands points to AhR also having a crucial role in normal cell physiology. Thus, AhR is emerging as a promiscuous receptor that can mediate either toxic or physiologic effects upon sensing multiple exogenous and endogenous molecules. Within this scenario, several factors appear to contribute to the outcome of gene transcriptional regulation by AhR, including the nature of the ligand as such and its further metabolism by AhR-induced enzymes, the local tissue microenvironment, and the presence of coregulators or specific transcription factors in the cell. Here, we review the current knowledge on the array of transcription factors and coregulators that, by interacting with AhR, tune its transcriptional activity in response to endogenous and exogenous ligands.

Duchenne muscular dystrophy is a lethal neuromuscular disorder caused by the absence of dystrophin, for which no curative treatment is available. RNA based approaches have shown promising results; however, their evaluation is hindered by the lack of robust and rapid delivery methods for differentiated human muscle cells, which represent the most physiologically relevant in vitro models for assessing therapeutic strategies. Here, we establish a versatile lipid nanoparticle platform enabling efficient delivery of diverse RNA therapeutics across a range of human muscle models, including myotubes, induced pluripotent stem cell-derived myotubes, myoblasts, cardiomyocytes, and 3D engineered skeletal muscle tissues. Remarkably, a single commercially available lipid nanoparticle formulation supports delivery of cargos spanning more than 300-fold in size, from short antisense oligonucleotides (~20 nt) to complex CRISPR-based editors (up to ~6.7 kb), including Cas9 nucleases, adenine base editors, and CRISPRa systems. This enables efficient gene correction and transcriptional modulation, resulting in dystrophin restoration or compensatory utrophin upregulation in relevant Duchenne muscular dystrophy models. Together, our results establish a single lipid nanoparticle formulation as a versatile platform for RNA delivery in human muscle systems and provide a practical framework for the rapid preclinical assessment of emerging therapies for Duchenne muscular dystrophy and other neuromuscular disorders.

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.