Crucial Mechanistic Insights Research Articles

Metabolites and Metabolism in Vascular Calcification: Links Between Adenosine Signaling and the Methionine Cycle.

The global population of individuals with cardiovascular disease is expanding, and a key risk factor for major adverse cardiovascular events is vascular calcification. The pathogenesis of cardiovascular calcification is complex and multifaceted, with external cues driving epigenetic, transcriptional, and metabolic changes that promote vascular calcification. This review provides an overview of some of the lesser understood molecular processes involved in vascular calcification and discusses the links between calcification pathogenesis and aspects of adenosine signaling and the methionine pathway; the latter of which salvages the essential amino acid methionine, but also provides the substrate critical for methylation, a modification that regulates the function and activity of DNA and proteins. We explore the complex and dynamic nature of osteogenic reprogramming underlying intimal atherosclerotic calcification and medial arterial calcification (MAC). Atherosclerotic calcification is more widely studied however emerging studies now show MAC is a significant pathology independent from atherosclerosis. Further, we emphasize metabolite and metabolic-modulating factors that influence vascular calcification pathogenesis. While the contribution of these mechanisms are more well-define in relation to atherosclerotic intimal calcification, understanding these pathways may provide crucial mechanistic insights into MAC and inform future therapeutic approaches. Herein we highlight the significance of adenosine and methyltransferase pathways as key regulators of vascular calcification pathogenesis.

Triiodide Formation Governs Oxidation Mechanism of Tin-Lead Perovskite Solar Cells via A-Site Choice.

Mixed tin-lead (Sn-Pb) halide perovskites stand out as promising materials for next-generation photovoltaics and near-infrared optoelectronics. However, their sensitivity to oxidative degradation remains a major hurdle toward their widespread deployment. A holistic understanding of their oxidation processes considering all their constituent ions is therefore essential to stabilize these materials. Herein, we reveal that A-site cation choice plays an inconspicuous yet crucial role in determining Sn-Pb perovskite stability toward oxidation. Comparing typical A-site compositions, we show that thin films and solar cells containing cesium are more resistant to oxidative stress relative to their methylammonium analogs. We identify degradation in these compositions to be closely linked to the presence of triiodide, a harmful species evolving from native I2 oxidants. We find that hydrogen bonding between methylammonium and I2 promotes triiodide formation, while the strong polarizing character of cesium limits this process by capturing I2. Inspired from these findings, we design two strategies to boost stability of sensitive methylammonium-based Sn-Pb perovskite films and devices against oxidation. Specifically, we modulate the polarizing character of surface A-sites in perovskite via CsI and RbI coatings, and we incorporate Na2S2O3 as an I2 scavenging additive. These crucial mechanistic insights will pave the way for the design of highly efficient and stable Sn-Pb perovskite optoelectronics.

Azobenzene-Based Photoswitchable Substrates for Advanced Mechanistic Studies of Model Haloalkane Dehalogenase Enzyme Family.

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

Enhanced structural stability and durability in lithium-rich manganese-based oxide via surface double-coupling engineering

Lithium-rich manganese-based oxides (LRMOs) exhibit high theoretical energy densities, making them a prominent class of cathode materials for lithium-ion batteries. However, the performance of these layered cathodes often declines because of capacity fading during cycling. This decline is primarily attributed to anisotropic lattice strain and oxygen release from cathode surfaces. Given notable structural transformations, complex redox reactions, and detrimental interface side reactions in LRMOs, the development of a single modification approach that addresses bulk and surface issues is challenging. Therefore, this study introduces a surface double-coupling engineering strategy that mitigates bulk strain and reduces surface side reactions. The internal spinel-like phase coating layer, featuring three-dimensional (3D) lithium-ion diffusion channels, effectively blocks oxygen release from the cathode surface and mitigates lattice strain. In addition, the external Li3PO4 coating layer, noted for its superior corrosion resistance, enhances the interfacial lithium transport and inhibits the dissolution of surface transition metals. Notably, the spinel phase, as excellent interlayer, securely anchors Li3PO4 to the bulk lattice and suppresses oxygen release from lattices. Consequently, these modifications considerably boost structural stability and durability, achieving an impressive capacity retention of 83.4% and a minimal voltage decay of 1.49 mV per cycle after 150 cycles at 1 C. These findings provide crucial mechanistic insights into the role of surface modifications and guide the development of high-capacity cathodes with enhanced cyclability.

Mechanistic insights into immune amnesia and immunity induced by measles infection and vaccination via comprehensive antiviral antibodies profiling

Abstract Measles is a disease known to induce acute immune amnesia and immunosuppression with increased susceptibility to other infections, while paradoxically conferring patients with life-long immunity to measles. However, it is not clear if the live-attenuated measles vaccine (LAMV) induces immunosuppression comparable to the wild-type measles (WT MeV) infection, nor how mechanistically LAMV and WT MeV affect antibodies specific to different pathogens. Therefore, we utilized VirScan, a phage-display immunoprecipitation and sequencing (PhIP-seq) technology for virome-wide detection of antibodies against viral epitopes, to compare longitudinal antibody diversity in plasma samples from WT MeV and LAMV-infected rhesus macaques (RMs) (n=10 and 6 respectively, with 5 longitudinal timepoints at D0, D14/15, D28, D84/89, D112/168/176). Preliminary analysis showed that WT MeV led to more long-term depletion of pre-existing antibodies to various viral peptides, while mounting a MeV-specific antibody response with greater intensity and breadth than LAMV-infected RM. Furthermore, in our sex difference analysis, male RMs elicited a stronger MeV response than female, particularly to N protein; also a more polyclonal MeV humoral response In summary, our work provided crucial mechanistic insights into immune amnesia and immunity induced by natural infection versus vaccination, highlighting the public health value of the measles vaccine in averting MeV-induced immune amnesia.

Structural Pharmacology of TRPV4 Antagonists.

The nonselective calcium-permeable Transient Receptor Potential Cation Channel Subfamily V Member4 (TRPV4)channel regulates various physiological activities. Dysfunction of TRPV4 is linked to many severe diseases, including edema, pain, gastrointestinal disorders, lung diseases, and inherited neurodegeneration. Emerging TRPV4 antagonists show potential clinical benefits. However, the molecular mechanisms of TRPV4 antagonism remain poorly understood. Here, cryo-electron microscopy (cryo-EM) structures of human TRPV4 are presented in-complex with two potent antagonists, revealing the detailed binding pockets and regulatory mechanisms of TRPV4 gating. Both antagonists bind to the voltage-sensing-like domain (VSLD) and stabilize the channel in closed states. These two antagonists induce TRPV4 to undergo an apparent fourfold to twofold symmetry transition. Moreover, it is demonstrated that one of the antagonists binds to the VSLD extended pocket, which differs from the canonical VSLD pocket. Complemented with functional and molecular dynamics simulation results, this study provides crucial mechanistic insights into TRPV4 regulation by small-molecule antagonists, which may facilitate future drug discovery targeting TRPV4.

Electrocatalytic Nitrate and Nitrite Reduction toward Ammonia Using Cu2O Nanocubes: Active Species and Reaction Mechanisms.

The electrochemical reduction of nitrate (NO3-) and nitrite (NO2-) enables sustainable, carbon-neutral, and decentralized routes to produce ammonia (NH3). Copper-based materials are promising electrocatalysts for NOx- conversion to NH3. However, the underlying reaction mechanisms and the role of different Cu species during the catalytic process are still poorly understood. Herein, by combining quasi in situ X-ray photoelectron spectroscopy (XPS) and operando X-ray absorption spectroscopy (XAS), we unveiled that Cu is mostly in metallic form during the highly selective reduction of NO3-/NO2- to NH3. On the contrary, Cu(I) species are predominant in a potential region where the two-electron reduction of NO3- to NO2- is the major reaction. Electrokinetic analysis and in situ Raman spectroscopy was also used to propose possible steps and intermediates leading to NO2- and NH3, respectively. This work establishes a correlation between the catalytic performance and the dynamic changes of the chemical state of Cu, and provides crucial mechanistic insights into the pathways for NO3-/NO2- electrocatalytic reduction.

Interaction analysis of SARS-CoV-2 omicron BA1 and BA2 of RBD with fifty monoclonal antibodies: Molecular dynamics approach

This report provides detailed insights into the interaction of fifty monoclonal antibodies with two recent Omicron variants, BA1 and BA2. It has been observed that numerous mutations in the receptor binding domain (RBD) result in significant structural changes in Omicron, enhancing its ability to mediate viral infections compared to other variants of concern. The following antibodies, namely JX3S304, 7KMG, 7CH4, 7BELCOVOX45, 7CDJ, 7C01, 7JX3S2H14, 6XCA, 7CDI, 7JMO, 7B3O, 6ZER, 6XC7CR3022, JX3S309, 6XC7CC123, 7CM4, 7KMI, 7L7EAZD8895, exhibit a superior binding affinity towards the Spike when compared to the reference CR3022. Four best-docked systems were subjected to further testing through molecular dynamics (MD) simulations. The MM/GBSA free energy for the top-scored complexes of BA1 variant are BA1_JX3S3O4, BA1_7KMI, BA1_7CH4, and BA1_7KMG, with respective values of −56.120 kcal/mol, −41.30 kcal/mol, −17.546 kcal/mol, and −8.527 kcal/mol; and of BA2 variant are BA2_JX3S3O4, BA2_7CM4, BA2_KMG, and BA2_7CH4, with respective values of −40.903 kcal/mol, −23.416 kcal/mol, −17.350 kcal/mol, and −5.460 kcal/mol. Detailed structural/energetic parameters, principal component analysis, and free energy landscape (FEL) studies reveal a significant decrease in antibody resistance due to the disappearance of numerous hydrogen bond interactions and various metastable states. We believe that these crucial mechanistic insights will contribute to breakthroughs in SARS-CoV-2 research.

Unveiling Synergistic Potency: Exploring Butyrolactone I to Enhance Gentamicin Efficacy against Methicillin-Resistant Staphylococcus aureus (MRSA) Strain USA300.

Staphylococcus aureus, including MRSA strains, poses significant health risks, imposing a significant disease burden and mortality. We investigate butyrolactone I (BL-1), a marine-derived metabolite from Aspergillus terreus, enhancing aminoglycoside efficacy against MRSA. A promising synergy is observed with BL-1 and various aminoglycosides, marked by low fractional inhibitory concentration indexes (FICIs < 0.5). Comprehensive studies utilizing USA300 MRSA and gentamicin reveal a remarkable one-fourth reduction in minimum inhibitory concentration (MIC) with 20 μg/mL BL-1. A relative abundance assay indicates that BL-1 enhances gentamicin uptake while restraining extracellular presence, involving intricate transmembrane signaling and molecular interactions. RNA-Seq analysis yielded an unexpected revelation, unveiling a distinctive gene expression profile and distinguishing it from other treatment approaches. Furthermore, meticulous analyses validated the extensive perturbations induced by BL-1 exposure, affecting diverse biological functions, encompassing glycolysis, amino acid metabolisms, substance transmembrane transport, and virulence generation. These valuable insights inspired further confirmation of bacterial virulence and the modulation of membrane permeability resulting from BL-1 treatment. Phenotypic validations corroborated our observations, revealing reduced membrane permeability and hemolytic toxicity, albeit demanding a deeper comprehension of the intricate interplay underlying these actions. Our study contributes crucial mechanistic insights to the development of therapeutic strategies against this notorious pathogen and the judicious employment of aminoglycosides. Additionally, it elucidates marine-derived metabolites' ecological and functional roles, exemplified by fungal quorum sensing signals. These compounds could give producers a competitive edge, inhibiting microorganism proliferation and suggesting novel approaches for combating resistant pathogens.

Transcriptomic analysis reveals upregulated host metabolisms and downregulated immune responses or cell death induced by acute African swine fever virus infection.

The African swine fever virus is a virulent and communicable viral disease that can be transmitted by infected swine, contaminated pork products, or soft tick vectors. Nonstructural proteins encoded by ASFV regulate viral replication, transcription, and evasion. However, the mechanisms underlying the host response to ASFV infection remain incompletely understood. In order to enhance comprehension of the biology and molecular mechanisms at distinct time intervals (6, 12, 24 h) post infection, transcriptome analyses were executed to discern differentially expressed genes (DEGs) between ASFV and mock-infected PAMs. The transcriptomic analysis unveiled a total of 1,677, 2,122, and 2,945 upregulated DEGs and 933, 1,148, and 1,422 downregulated DEGs in ASFV- and mock-infected groups at 6, 12, and 24 h.p.i.. The results of the transcriptomic analysis demonstrated that the infection of ASFV significantly stimulated host metabolism pathways while concurrently inhibiting the expression of various immune responses and cell death pathways. Our study offers crucial mechanistic insights into the comprehension of ASFV viral pathogenesis and the multifaceted host immune responses. The genes that were dysregulated may serve as potential candidates for further exploration of anti-ASFV strategies.

AMPK–FOXO–IP3R signaling pathway mediates neurological and developmental defects caused by mitochondrial DNA mutations

Pathological mutations in human mitochondrial genomes (mtDNA) can cause a series of neurological, behavioral, and developmental defects, but the underlying molecular mechanisms are poorly understood. We show here that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway plays a key role in mediating similar defects caused by different mtDNA mutations in Caenorhabditis elegans, including loss or reduction of osmotic, chemical and olfactory sensing, locomotion, and associative learning and memory, as well as increased embryonic lethality. mtDNA mutations cause reduced ATP (adenosine triphosphate) levels, activation of C. elegans AMPK AAK-2, and nuclear translocation of the FOXO transcription factor DAF-16. Activated DAF-16 up-regulates the expression of inositol triphosphate receptor ITR-1, an endoplasmic reticulum calcium channel, leading to increased basal cytosolic Ca2+ levels, decreased neuronal responsiveness, compromised synapses, and increased embryonic death. Treatment of mtDNA mutants with vitamin MK-4 restores cellular ATP and cytosolic Ca2+ levels, improves synaptic development, and suppresses sensory and behavioral defects and embryonic death. Our study provides crucial mechanistic insights into neuronal and developmental defects caused by mtDNA mutations and will improve understanding and treatment of related mitochondrial diseases.

Interrogating the substrate specificity landscape of UvrC reveals novel insights into its non-canonical function

Although it is relatively unexplored, accumulating data highlight the importance of tripartite crosstalk between nucleotide excision repair (NER), DNA replication, and recombination in the maintenance of genome stability; however, elucidating the underlying mechanisms remains challenging. While Escherichia coli uvrA and uvrB can fully complement polAΔ cells in DNA replication, uvrC attenuates this alternative DNA replication pathway, but the exact mechanism by which uvrC suppresses DNA replication is unknown. Furthermore, the identity of bona fide canonical and non-canonical substrates for UvrCs are undefined. Here, we reveal that Mycobacterium tuberculosis UvrC (MtUvrC) strongly binds to, and robustly cleaves, key intermediates of DNA replication/recombination as compared with the model NER substrates. Notably, inactivation of MtUvrC ATPase activity significantly attenuated its endonuclease activity, thus suggesting a causal link between these two functions. We built an in silico model of the interaction of MtUvrC with the Holliday junction (HJ), using a combination of homology modeling, molecular docking, and molecular dynamic simulations. The model predicted residues that were potentially involved in HJ binding. Six of these residues were mutated either singly or in pairs, and the resulting MtUvrC variants were purified and characterized. Among them, residues Glu595 and Arg597 in the helix-hairpin-helix motif were found to be crucial for the interaction between MtUvrC and HJ; consequently, mutations in these residues, or inhibition of ATP hydrolysis, strongly abrogated its DNA-binding and endonuclease activities. Viewed together, these findings expand the substrate specificity landscape of UvrCs and provide crucial mechanistic insights into the interplay between NER and DNA replication/recombination.

Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro.

Hepadnaviruses, including hepatitis B virus (HBV) as a major human pathogen, replicate their tiny 3 kb DNA genomes by capsid-internal protein-primed reverse transcription of a pregenomic (pg) RNA. Initiation requires productive binding of the viral polymerase, P protein, to a 5´ proximal bipartite stem-loop, the RNA encapsidation signal ε. Then a residue in the central ε bulge directs the covalent linkage of a complementary dNMP to a Tyr sidechain in P protein´s Terminal Protein (TP) domain. After elongation by two or three nucleotides (nt) the TP-linked DNA oligo is transferred to a 3´ proximal acceptor, enabling full-length minus-strand DNA synthesis. No direct structural data are available on hepadnaviral initiation complexes but their cell-free reconstitution with P protein and ε RNA (Dε) from duck HBV (DHBV) provided crucial mechanistic insights, including on a major conformational rearrangement in the apical Dε part. Analogous cell-free systems for human HBV led at most to P—ε binding but no detectable priming. Here we demonstrate that local relaxation of the highly basepaired ε upper stem, by mutation or via synthetic split RNAs, enables ε-dependent in vitro priming with full-length P protein from eukaryotic translation extract yet also, and without additional macromolecules, with truncated HBV miniP proteins expressed in bacteria. Using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) we confirm that upper stem destabilization correlates with in vitro priming competence and show that the supposed bulge-closing basepairs are largely unpaired even in wild-type ε. We define the two 3´ proximal nt of this extended bulge as main initiation sites and provide evidence for a Dε-like opening of the apical ε part upon P protein binding. Beyond new HBV-specific basic aspects our novel in vitro priming systems should facilitate the development of high-throughput screens for priming inhibitors targeting this highly virus-specific process.

Embracing human genetics: a primer for developmental biologists.

Understanding the etiology of congenital disorders requires interdisciplinary research and close collaborations between clinicians, geneticists and developmental biologists. The pace of gene discovery has quickened due to advances in sequencing technology, resulting in a wealth of publicly available sequence data but also a gap between gene discovery and crucial mechanistic insights provided by studies in model systems. In this Spotlight, I highlight the opportunities for developmental biologists to engage with human geneticists and genetic resources to advance the study of congenital disorders.

A Pulsatile Flow System to Engineer Aneurysm and Atherosclerosis Mimetic Extracellular Matrix.

Alterations of blood flow patterns strongly correlate with arterial wall diseases such as atherosclerosis and aneurysm. Here, a simple, pumpless, close‐loop, easy‐to‐replicate, and miniaturized flow device is introduced to concurrently expose 3D engineered vascular smooth muscle tissues to high‐velocity pulsatile flow versus low‐velocity disturbed flow conditions. Two flow regimes are distinguished, one that promotes elastin and impairs collagen I assembly, while the other impairs elastin and promotes collagen assembly. This latter extracellular matrix (ECM) composition shares characteristics with aneurysmal or atherosclerotic tissue phenotypes, thus recapitulating crucial hallmarks of flow‐induced tissue morphogenesis in vessel walls. It is shown that the mRNA levels of ECM of collagens and elastin are not affected by the differential flow conditions. Instead, the differential gene expression of matrix metalloproteinase (MMP) and their inhibitors (TIMPs) is flow‐dependent, and thus drives the alterations in ECM composition. In further support, treatment with doxycycline, an MMP inhibitor and a clinically used drug to treat vascular diseases, halts the effect of low‐velocity flow on the ECM remodeling. This illustrates how the platform can be exploited for drug efficacy studies by providing crucial mechanistic insights into how different therapeutic interventions may affect tissue growth and ECM assembly.

Probing Mechanisms of Transcription Elongation Through Cell-to-Cell Variability of RNA Polymerase

The process of transcription initiation and elongation are primary points of control in the regulation of gene expression. Although biochemical studies have uncovered the mechanisms involved in controlling transcription at each step, how these mechanisms manifest in vivo at the level of individual genes is still unclear. Recent experimental advances have enabled single-cell measurements of RNA polymerase (RNAP) molecules engaged in the process of transcribing a gene of interest. In this article, we use Gillespie simulations to show that measurements of cell-to-cell variability of RNAP numbers and interpolymerase distances can reveal the prevailing mode of regulation of a given gene. Mechanisms of regulation at each step, from initiation to elongation dynamics, produce qualitatively distinct signatures, which can further be used to discern between them. Most intriguingly, depending on the initiation kinetics, stochastic elongation can either enhance or suppress cell-to-cell variability at the RNAP level. To demonstrate the value of this framework, we analyze RNAP number distribution data for ribosomal genes in Saccharomyces cerevisiae from three previously published studies and show that this approach provides crucial mechanistic insights into the transcriptional regulation of these genes.

The effect of amantadine on an ion channel protein from Chikungunya virus.

Viroporins like influenza A virus M2, hepatitis C virus p7, HIV-1 Vpu and picornavirus 2B associate with host membranes, and create hydrophilic corridors, which are critical for viral entry, replication and egress. The 6K proteins from alphaviruses are conjectured to be viroporins, essential during egress of progeny viruses from host membranes, although the analogue in Chikungunya Virus (CHIKV) remains relatively uncharacterized. Using a combination of electrophysiology, confocal and electron microscopy, and molecular dynamics simulations we show for the first time that CHIKV 6K is an ion channel forming protein that primarily associates with endoplasmic reticulum (ER) membranes. The ion channel activity of 6K can be inhibited by amantadine, an antiviral developed against the M2 protein of Influenza A virus; and CHIKV infection of cultured cells can be effectively inhibited in presence of this drug. Our study provides crucial mechanistic insights into the functionality of 6K during CHIKV-host interaction and suggests that 6K is a potential therapeutic drug target, with amantadine and its derivatives being strong candidates for further development.

Disruption of Long Noncoding RNAs Targets Cancer Hallmark Pathways in Lung Tumorigenesis.

Advances in high-throughput genomic and epigenomic technologies have revealed the tremendous complexity of the transcriptional landscape. Beyond protein-coding RNAs (derived from only ∼1.5% of the genome), noncoding RNAs (ncRNA) are emerging as versatile key regulators of gene information involved in multiple major biological processes. Accordingly, deregulation of ncRNA expression has been associated with multiple diseases, including cancer. In this issue of Cancer Research, Shahabi and colleagues characterize LINC00261 as a tumor suppressor long ncRNA epigenetically silenced in lung cancer. They provide crucial mechanistic insights to explain its role in lung tumorigenesis, demonstrating that deregulation of the LINC00261/FOXA2 locus disrupts DNA damage repair signaling, cell-cycle control, and cell proliferation.See related article by Shahabi et al., p. 3050.

Visualizing biologically active small molecules in cells using click chemistry

Natural products and synthetic small molecules can be used to perturb, dissect and manipulate biological processes, thereby providing the basis for drug development. Over the past decades, the evolution of molecular biology protocols and microscopy techniques has made it possible to visually detect proteins in living systems with valuable spatiotemporal resolution, in which dynamic topological information has proved to be insightful. By contrast, although small molecules have become essential for biological studies, general methods to track them in cells remain underexplored. In this Review, we discuss how bioorthogonal chemistry, and click chemistry in particular, can be exploited to label and visualize almost any biologically active small molecule in cells and tissues. We review recent developments, highlighting cases in which visualizing small molecules has provided crucial mechanistic insights. This methodology is facile to implement, is versatile and is illuminating. Click chemistry enables efficient chemical labelling of small molecules in cells, providing a powerful method to visualize almost any biologically active compound. This versatile methodology can provide valuable information about the mechanisms of action of small molecules in various biological settings.

CD122 blockade restores immunological tolerance in autoimmune type 1 diabetes via multiple mechanisms.

Signaling through IL-2/IL-15Rβ (CD122) is essential for the differentiation and function of T cells and NK cells. A mAb against CD122 has been implicated to suppress autoimmune type 1 diabetes (T1D) development in animal models. However, the mechanisms remain poorly understood. We find that in vivo administration of an anti-CD122 mAb (CD122 blockade) restores immune tolerance in nonobese diabetic (NOD) mice via multiple mechanisms. First, CD122 blockade selectively ablates pathogenic NK cells and memory phenotype CD8+ T cells from pancreatic islets. In contrast, islet CD4+Foxp3+ Tregs are only mildly affected. Second, CD122 blockade suppresses IFN-γ production in islet immune cells. Third, CD122 blockade inhibits the conversion of islet Th17 cells into diabetogenic Th1 cells. Furthermore, a combination of anti-CD122 mAb and Treg-trophic cytokines (IL-2 or IL-33) enhances the abundance and function of islet Tregs. In summary, these data provide crucial mechanistic insights into CD122 blockade-mediated immunoregulation and support therapeutic benefits of this combinational treatment in T1D.