Biology subjects

Irani, S.

Publications and source records attributed to Irani, S..

10 recordsLinked to original sources

bioRxiv2026

Antibodies with Engineered Fc Domains Having Absolute Binding Selectivity to either FcγRIIa or FcγRI Delineate the Respective Effector Phenotypes by Human Monocytes and Macrophages

IgG1 immune complexes bind to all the Fc{gamma} receptors (Fc{gamma}R) expressed on myeloid cells, making it challenging to determine the precise role of each Fc{gamma}R on Fc effector phenotypes. Here we report the engineering of Fc2KG, an aglycosylated human IgG1 Fc domain that binds with near physiological affinity to Fc{gamma}RIIa/b with no detectable binding to any other Fc{gamma}Rs. Crystallographic analysis elucidated the structural basis of how mutations in the Fc domain compensate for the absence of the N297 glycan and enable selective binding. Using single-cell phagocytosis assays, we show that particles opsonized with Fc-engineered antibodies formatted with Fc2KG or with an Fc domain that binds only Fc{gamma}RI (Fc5), are ingested by THP-1 cells with near identical kinetics. We find that with CD16+ primary human monocytes, selective Fc{gamma}RII engagement is a major contributor to the ADCP mediated by wild-type IgG1. Additionally, we showed that Fc2KG formatted antibodies induce high levels of GM-CSF. With M1-like monocyte-derived human macrophages, trastuzumab formatted with wild-type IgG1 Fc, with Fc2KG or Fc5 were all equally proficient in the trogocytotic killing of opsonized SK-BR-3 HER2+ cells but only Fc{gamma}RI engagement led to secretion of proinflammatory cytokines. Collectively, our results highlight how precisely tuned, Fc-engineered antibodies can be deployed to answer long-standing questions regarding the precise effector functions mediated by human Fc{gamma}Rs, information which is key for the optimization of therapeutic antibodies.

immunology↗

bioRxiv2025

Combinatorial phosphorylation on CTD of RNA polymerase II selectively controls transcription and export of protein-coding mRNAs

C-terminal domain (CTD) of RNA polymerase II is crucial for recruiting transcription regulators via specific post-translational modifications (PTM), especially phosphorylation. The hypothesis of combination of PTMs, or CTD code, that can allow precise and dynamic recruitment of transcription machinery is highly attractive, yet the experimental evidence to support this hypothesis has been scarce. Here, despite lacking specific antibodies for combinatorial CTD phosphorylation, we developed an innovative approach that detects double phosphorylation patterns on the CTD in a whole-genomic fashion by leveraging the antibody masking effect with selectively removing the flanking interference. Using this method, we detected pT4pS5 double phosphosites occurring exclusively during the transcription of protein-coding genes. Furthermore, we showed that pT4pS5 marks recruit the Transcription and Export complex (TREX), which specifically facilitates mRNA processing and nucleocytoplasmic export of protein-coding mRNAs. The recruitment of TREX by pT4pS5 phosphosites is particularly important for the processing of lengthy neurogenesis-related genes. Our results provide experimental support for the notion that CTD coding system can function combinatorially and in a gene-specific manner, which encodes an exact information about the transcription of specific gene clusters. This method can be broadly applied to map all combinatorial PTM patterns on RNA polymerase II, paving the way for a deeper understanding of gene-specific transcription regulation at the molecular level.

molecular biology↗

bioRxiv2025

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

bioRxiv2024

Neurodegenerative fluid biomarkers are enriched in human cervical lymph nodes

In animal models, brain neurodegeneration biomarkers drain into cervical lymph nodes (CLNs). If this occurred in humans, CLNs may provide a readily accessible source of these biomarkers, draining the site of primary pathology. We tested this hypothesis in discovery and validation cohorts using ultrasound-guided fine needle aspiration (FNA). We measured amyloid-beta 40 and 42, phospho-Tau-181, glial-fibrillary-acidic-protein, and neurofilament-light using single molecule array in CLN aspirates and plasma from: i) a discovery cohort of 25 autoimmune patients, and from ii) plasma, CLNs and capillary blood in four healthy volunteers, an optimisation-validation cohort. FNA was well-tolerated by all participants. In both cohorts, all biomarkers were detected in all plasmas and CLNs, other than neurofilament-light (8/17 of discovery cohort). CLN biomarker concentrations were significantly greater than plasma concentrations for all except neurofilament-light, most markedly for phospho-Tau-181 (266 fold; P<0.02), whose CLN concentrations decreased with age (Spearman r=-0.66, P=0.001). This study presents the first evidence that neurodegenerative biomarkers are detectable in human CLNs. Raised CLN:plasma biomarker ratios suggest their concentration in CLNs, which may offer a sensitive compartment for minimally-invasive sampling in clinical trials. Further, age-associated phospho-Tau-181 reduction with age suggests FNA of CLNs may measure the integrity of brain lymphatic drainage in vivo.

neuroscience↗

bioRxiv2023

Peripherally derived LGI1-reactive monoclonal antibodies cause epileptic seizures in vivo

One striking clinical hallmark in patients with autoantibodies to leucine-rich glioma inactivated 1 (LGI1) is the very frequent focal seizure semiologies, including faciobrachial dystonic seizures (FBDS), in addition to the amnesia. Polyclonal serum IgGs have successfully modelled the cognitive changes in vivo but not seizures. Hence, it remains unclear whether LGI1-autoantibodies are sufficient to cause seizures. We tested this with the molecularly precise monoclonal antibodies directed against LGI1 (LGI1-mAbs), derived from patient circulating B cells. These were directed towards both major domains of LGI1, LRR (n=5) and EPTP (n=5) and infused intracerebroventricularly over 7 days into juvenile male Wistar rats using osmotic pumps. Continuous wireless EEG was recorded from a depth electrode placed in hippocampal CA3 plus behavioural tests for memory and hyperexcitability were performed. Following infusion completion (Day 9), post-mortem brain slices were studied using electrophysiology and immunostaining. By comparison to control-mAb injected rats (n=6), video-EEG analysis over 9 days revealed convulsive and non-convulsive seizure activity in rats infused with LGI1-mAbs, with a significant number of ictal events (245{+/-}83 vs. 7.8{+/-}7.8 in controls; p=0.002). Memory was not impaired in the novel object recognition test. Local field potential recordings from postmortem brain slices showed spontaneous ictal-like spike activity in the CA3 region (p=0.03). The LGI1-mAbs bound most strongly in the hippocampal CA3 region and induced a significant reduction in Kv1.1 cluster number in this subfield (6 controls; 7 LGI1-mAbs; p=0.01) Peripherally-derived human LGI1-mAbs infused into rodent CSF provide strong evidence of direct in vivo epileptogenesis with molecular correlations. These findings fulfill criteria for LGI1-antibodies in seizure causation.

neuroscience↗

bioRxiv2021

The SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we used domain-focused CRISPR screening to identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

cancer biology↗

bioRxiv2020

Lipidomic profiling of clinical prostate cancer reveals targetable alterations in lipid composition

Dysregulated lipid metabolism is a prominent feature of prostate cancer that is driven by androgen receptor (AR) signaling. Herein, we used quantitative mass spectrometry to define the "lipidome" in prostate tumors with matched benign tissues (n=21), independent tissues (n=47), and primary prostate explants cultured with a clinical AR antagonist, enzalutamide (n=43). Significant differences in lipid composition were detected and spatially visualized in tumors compared to matched benign samples. Notably, tumors featured higher proportions of monounsaturated lipids overall and elongated fatty acid chains in phosphatidylinositol and phosphatidylserine lipids. Significant associations between lipid profile and malignancy were validated in unmatched samples, and PL composition was characteristically altered in patient tissues that responded to AR inhibition. Importantly, targeting of altered tumor-related lipid features, via inhibition of acetyl CoA carboxylase 1, significantly reduced cellular proliferation in tissue explants (n=13). This first characterization of the prostate cancer lipidome in clinical tissues revealed enhanced fatty acid synthesis, elongation and desaturation as tumor-defining features, with potential for therapeutic targeting.

cancer biology↗

bioRxiv2020

A feedback loop between the androgen receptor and 6-phosphogluoconate dehydrogenase (6PGD) drives prostate cancer growth

Alterations to androgen receptor (AR) signalling and cellular metabolism are hallmarks of prostate cancer. This study uncovers a novel link between AR and the pentose phosphate pathway (PPP) through 6-phosphogluoconate dehydrogenase (6PGD), an androgen-regulated gene that is upregulated in prostate cancer. Knockdown of 6PGD impairs growth and elicits death of prostate cancer cells, at least in part due to oxidative stress. Targeting 6PGD using 2 specific inhibitors, physcion and S3, was efficacious in multiple models of prostate cancer, including aggressive castration-resistant models. Importantly, S3 also suppressed proliferation of clinical patient-derived explants (PDEs). Mechanistically, 6PGD decreased expression and activity of AR in cell lines and PDEs, revealing a novel positive feedback loop between these factors. The enhanced efficacy of co-targeting AR and 6PGD further supported the biological relevance of this feedback. This work provides insight into the dysregulated metabolism of prostate cancer and supports investigation of co-targeting AR and the PPP.

cancer biology↗

bioRxiv2019

DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis

Fatty acid {beta}-oxidation (FAO) is the main bioenergetic pathway in prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify DECR1, which encodes the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown in PCa cells selectively inhibited {beta}-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation in vivo. Mechanistically, targeting of DECR1 caused cellular accumulation of linoleic acid, enhanced mitochondrial oxidative stress and lipid peroxidation, and ferroptosis. These findings implicate PUFA oxidation via DECR1 as a previously unexplored facet of FAO that promotes survival of PCa cells.

cancer biology↗

bioRxiv2019

Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase IIs C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes a reduction of phosphorylated Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTDs coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

biochemistry↗