Journal of Clinical Investigation

AbstractNon-obstructive azoospermia (NOA) is a severe form of male infertility. Proteins from the Tre2-Bub2-Cdc16 (TBC) family emerge as key contributors to spermatogenesis, including TBC1D20, TBC1D21, and TBC1D25. Nonetheless, the specific role of TBC1D8 in male male fertility remains unclear. Our study aims to elucidate the relationship between TBC1D8 and male infertility in humans and mice. Male Tbc1d8-/- mice were completely infertile, producing no offspring. They exhibited spermiogenesis defects from steps 9 to 16, accompanied by pronounced acrosomal abnormalities during sperm maturation, ultimately preventing the formation of functional spermatozoa. Mechanistically, TBC1D8 deficiency impaired autophagic flux and enhanced apoptosis in the seminiferous epithelium through disrupted coupling with RAB8A, culminating in azoospermia and infertility. Consistent with the murine phenotype, whole-exome sequencing (WES) identified compound heterozygous missense variants in TBC1D8 (NM_001102426.3) among three patients from unrelated Chinese Han families diagnosed with NOA or cryptozoospermia: Patient 1: c.3322A>G and c.1747C>T; Patient 2: c.3322A>G and c.2566A>G; Patient 3: c.845C>T and c.2525C>T. Histological examination of patient testicular biopsies revealed severely disrupted spermatogenic architecture and markedly reduced TBC1D8 expression, suggesting a loss-of- function effect. Together, our findings establish TBC1D8 as a previously unrecognized regulator of spermatogenesis, where its deficiency leads to NOA through impaired autophagy and increased apoptosis..

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm nucleus. The physiological roles of PT in sperm biology and the clinical relevance of variants of PT proteins to male infertility are still largely unknown. We reveal that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts with itself, several other PT proteins, the inner acrosomal membrane (IAM) protein SPACA1, and the nuclear envelope (NE) protein FAM209 to form an IAM-cylicins-NE sandwich structure, anchoring the acrosome to the nucleus. WES of more than 500 Chinese infertile men with sperm head deformities was performed and a CYLC1 variant was identified in 19 patients. Cylc1-mutant mice carrying this variant also exhibited sperm acrosome/head deformities and reduced fertility, indicating that this CYLC1 variant most likely affects human male reproduction. Furthermore, the outcomes of assisted reproduction were reported for patients harbouring the CYLC1 variant. Our findings demonstrate a critical role of cylicin-1 in the sperm acrosome-nucleus connection and suggest CYLC1 variants as potential risk factors for human male fertility.

The loss of lysosomal cystine transporter cystinosin (CTNS) disrupts kidney proximal tubule (PT) function, causing cystinosis - a prototypical lysosomal storage disorder characterized by cystine accumulation and metabolic dyshomeostasis. Cystine storage disrupts lysosomal nutrient sensing and downstream mTORC1 signalling, driving loss of PT differentiation and proximal tubulopathy. Here, using cross-species disease models, differentiated cellular systems, lysosome-based assays, and transcriptomics profiling, we demonstrate that sodium-glucose co-transporter 2 (SGLT2) inhibitors (empagliflozin or dapagliflozin) ameliorate proximal tubulopathy in cystinosis. In CTNS-deficient PT cells, SGLT2 inhibition restores lysosome proteolysis, autophagic flux, metabolic homeostasis, and epithelial differentiation and function, independently of cystine clearance. Mechanistically, SGLT2 inhibition reduces the assembly of the v-ATPase-Ragulator-Rag scaffolding complex at lysosomes, thereby decoupling cystine storage from pathological mTORC1 activation. These effects reprogram PT metabolic trajectories and differentiation states, mitigating proximal tubulopathy across zebrafish and rodent models of cystinosis. Together, these findings define a lysosome-metabolism crosstalk that links glucose handling to mTORC1 regulation and provide a rationale for repurposing SGLT2 inhibitors as a disease-modifying therapy for cystinosis and related lysosome-driven PT disorders.

Symmetric, progressive, necrotizing lesions in the brainstem are a defining feature of Leigh syndrome (LS). A mechanistic understanding of the pathogenesis of these lesions has been elusive. Here, we report that leukocyte proliferation is causally involved in the pathogenesis of Leigh syndrome. Directly depleting leukocytes with a colony-stimulating factor 1 receptor (CSF1R) inhibitor dramatically attenuates disease, including complete prevention of CNS lesion formation and substantial extension of survival. Leukocyte depletion rescues a range of symptoms including hyperlactemia, seizures, respiratory function, and neurologic symptoms. These data provide a mechanistic explanation for the beneficial effects of mTOR inhibition. More importantly, these findings dramatically alter our understanding of the pathogenesis of LS, demonstrating that immune involvement directly drives disease. These findings have significant implication for the mechanisms of disease resulting from mitochondrial dysfunction, and may lead to novel therapeutic strategies. One-Sentence SummaryPharmacologic targeting of leukocytes prevents CNS lesions, neurological disease, and metabolic dysfunction in the Ndufs4(KO) mouse model of Leigh syndrome.

Endometriosis is a common chronic gynecological disorder with complex and poorly understood etiology and no definitive cure. Beyond pain and infertility, affected individuals frequently experience psychiatric comorbidities, often attributed to the burden of chronic symptoms. Emerging evidence suggests this explanation is incomplete, and shared biological mechanisms may also contribute. Here, we integrate large-scale genomic data to characterize the genetic overlap between endometriosis and a broad spectrum of psychiatric conditions. We found no evidence that genetic liability to endometriosis increases the risk of psychiatric conditions. In contrast, genetic liability to psychiatric conditions, particularly major depressive disorder and related traits, was associated with increasing the risk of endometriosis. Polygenic analyses revealed extensive shared genetic architecture, with nearly all variants influencing endometriosis also implicated in depression. Leveraging this overlap in a multivariate GWAS, we identify 606 independent genome-wide significant variants contributing to shared liability and implicate convergent biological pathways - particularly brain-related mechanisms - providing a foundation for mechanistic studies and potential strategies to mitigate psychiatric comorbidity in endometriosis.

Cystinuria is a rare inherited disease characterized by increased urinary cystine levels resulting in the formation of cystine stones in the urinary tract. Mutations in the genes encoding the cystine transporter complex, SLC3A1 and SLC7A9, are the primary drivers of the disease. Current mouse models used to study cystinuria rely on gene deficiency or spontaneous mutations in mice that do not accurately reflect the pathogenic mutations found in humans. To overcome this limitation, we generated novel Slc7a9G105R mice carrying the most common pathogenic single-point mutation in the SLC7A9 gene. Both male and female Slc7a9G105R mice developed a cystinuria phenotype by 9 weeks of age, characterized by substantial cystine stone formation and increased urinary cystine, lysine, arginine, and ornithine. Slc7a9G105R mice displayed distinct serum and urinary metabolite profiles mapped to dibasic amino acid pathways. Fecal metagenomics revealed that Slc7a9G105R mice had a heterogeneous microbiota with altered functional pathways, including increased L-cysteine biosynthesis. Depletion of the microbiota with antibiotics did not impact cystine stone burden but reduced urinary tract inflammation. Prophylactic or therapeutic dietary supplementation with alpha-lipoic acid reduced stone burden and inflammation, but it also caused damage to the urothelium. Untargeted metabolomics analysis following alpha-lipoic acid supplementation identified metabolites that can increase cystine solubility, reduce inflammation, and damage epithelial cells. Correlation analysis revealed novel serum biomarkers of stone burden, including blood urea nitrogen, 2-hydroxybutyric acid, 2-amino-2-thiazoline-4-carboxylic acid, and indole-3-acetylglycine. Collectively, the Slc7a9G105R mutant mouse model offers a precise, rapid-onset, and translational platform for investigating cystinuria pathogenesis and evaluating potential therapeutic strategies. Translational statementThe development of a novel knock-in mouse model carrying the most common pathogenic point mutation in the human SLC7A9 gene provides a clinically relevant and translationally valuable platform for investigating cystinuria pathogenesis and testing emerging therapies. This model represents the closest possible approximation of human SLC7A9-mediated cystinuria, enabling rigorous preclinical evaluation of small molecules and gene therapies. It has also facilitated the identification of candidate biomarkers for cystine stone burden and treatment response, which are urgently needed to improve disease monitoring and clinical decision-making. The next critical step is to validate these biomarkers in human cystinuria cohorts to support their clinical translation.

Despite high rates of post-surgical recurrence in men with high-risk localized prostate cancer (PCa), there is currently no role for neoadjuvant therapy. Tumor infiltrating regulatory T cells (TI-Tregs) limit the antitumor effects of presurgical androgen deprivation therapy (ADT). Therefore, we designed a neoadjuvant clinical trial to test whether Treg depletion via a non-fucosylated anti-CTLA-4 antibody (BMS-986218) is feasible and augments response to ADT. In this single-center, two-arm, open-label study, 24 men with high-risk localized PCa were randomized to ADT with or without BMS-986218 prior to radical prostatectomy. Treatment was well tolerated and feasible. Mechanistic studies indicated BMS-986218 depleted TI-Tregs by engaging CD16a/FCGR3A on tumor macrophages, modulated dendritic cells (DCs), and augmented T cell priming. Depth of Treg depletion and increased DC frequencies were quantitatively associated with improved clinical outcome. Overall, this study supports the feasibility and biological activity of neoadjuvant immunotherapy with ADT + Fc- enhanced anti-CTLA-4 in high-risk localized PCa. Statement of SignificanceNext-generation antibodies targeting CTLA-4 have been engineered for enhanced tumor Treg depletion in patients, yet their mechanisms of action remain incompletely defined. We performed the first single cell multi-omic correlative analyses of response to a glycoengineered anti-CTLA-4 antibody and defined mechanisms associated with clinical outcome in patients with high-risk localized prostate cancer.

Gaucher disease, an inborn error of glucosylceramide recycling, predisposes to haematological malignancies, liver cirrhosis and cancer. Inducible Gaucher disease modelled in susceptible mouse strains showed striking autoimmune hepatitis and cancer development; as reported previously, they also develop monoclonal immunoglobulins and B-cell tumours. Exposure to eliglustat, a therapeutic inhibitor of glucosylceramide biosynthesis, suppressed hepatic disease and occurrence of monoclonal immunoglobulins. To interrogate activation of CD1-restricted T cells by glucosylceramides, we deleted CD1d1/Cd1d2 function in the conditional inducible murine model of Gaucher disease with GBA1 deficiency in haematopoietic cells. Systemic inflammation and autoantibodies in these CD1-/- GCflox/flox Cre+ mice were markedly reduced, and severe liver injury and tumour formation was suppressed; however, occurrence of monoclonal immunoglobulins was unaffected. We suggest that CD1 molecules have distinct actions on tissue homeostasis in this system related to cell-intrinsic activation as well as glycolipid loading and presentation. Our findings implicate glycosphingolipids in the development of autoimmunity and cancer in human Gaucher disease and have broader significance in acquired and genetic disorders affecting sphingolipid expression.

Male infertility is associated with elevated rates of aneuploidy and DNA breaks in spermatozoa and germline precursors. This common condition is not well understood and is associated with poor individual and familial somatic health relative to fertile men. To further understand the extent and source of genome instability, we used error-corrected duplex DNA sequencing to test whether the impaired spermatogenesis and relatively poorer health of oligozoospermic men are linked to elevated single nucleotide de novo mutation frequencies in their sperm and blood, respectively. We observed a significant 1.34 to 2.01-fold increase in age-adjusted sperm mutation frequencies in infertile, oligozoospermic men. Conversely, consistently elevated mutation frequencies in the blood of oligozoospermic men were not found. Gain-of-function mutations linked to clonal spermatogenesis and Mendelian disorders accumulate with age at a similar rate in normozoospermic and oligozoospermic men. These results implicate germline hypermutation as a hallmark feature of oligozoospermia and point to age-independent processes affecting spermatogonial stem cell biology that may underlie spermatogenic impairment before and after puberty. Our findings also underscore the importance of investigating tissue-specific mechanisms driving the association between reduced reproductive and somatic health in infertile men.

Dysfunction of podocytes, cells critical for glomerular filtration, underlies proteinuria and kidney failure. Genetic forms of proteinuric kidney disease can be caused by mutations in several podocyte genes, including nephrin, a critical component of the kidney filter. In contrast, the etiology of acquired acute-onset nephrotic syndrome has remained elusive. Here we identify autoantibodies against nephrin in serum and glomeruli of a subset of adults and children with non-congenital acute nephrotic syndrome. Our findings align with published experimental animal studies and elucidate a novel autoimmune phenomenon in proteinuric kidney disease interfering with glomerular filter integrity.

Carbamazepine (CBZ) is a widely used antiepileptic drug effective in managing partial and generalized tonic-clonic seizures. Despite its established therapeutic efficacy, CBZ has been reported to worsen seizures in another form of epilepsy, generalized absence seizures, in both clinical and experimental settings. In this study, we focused on thalamic reticular (RT) neurons, which regulate thalamocortical network activity in absence seizures, to investigate whether CBZ alters their excitability, thereby contributing to the exacerbation of seizures. Using ex vivo whole-cell patch-clamp electrophysiology, we found that CBZ selectively inhibits the tonic firing of RT neurons in a dose-dependent manner without affecting burst firing. At the RT-thalamocortical (RT-TC) synapse, CBZ significantly increases the failure rate of GABAergic synaptic transmission, with greater effects on somatostatin (SST) - than parvalbumin (PV) - expressing RT neurons. In vivo EEG recordings and open-field behavior in Scn8amed+/- mouse model confirmed that CBZ treatment exacerbates absence seizures, increasing both seizure frequency and duration while reducing locomotor activity. In addition, CBZ further amplifies the pre-existing reduction in tonic firing of RT in Scn8amed+/- mice. These findings uncover a novel mechanism by which CBZ exacerbates absence seizures through selective inhibition of RT neuron excitability and disruption of GABAergic synaptic transmission. This work provides mechanistic insights into the paradoxical effects of CBZ and suggest potential avenues for optimizing epilepsy treatment strategies. Scientific SignificanceThis study addresses the clinical paradox in which CBZ, a widely prescribed antiepileptic drug, paradoxically aggravates absence seizures. Understanding the cellular mechanisms behind this phenomenon is critical for improving epilepsy treatments. Here, using electrophysiology recordings from intact thalamocortical slices and SCN8amed+/- mice, an absence seizure animal model, we demonstrate that CBZ selectively inhibits tonic firing of RT neurons and their output to thalamocortical circuits, with a more pronounced effect in SCN8amed+/- mice. These novel findings provide a mechanistic explanation for CBZs paradoxical aggravation of absence seizures, offering a framework for understanding the pharmacological effects of other anti-epilepsy drugs and guiding the development of more effective therapeutic strategies for epilepsy.

Red blood cells (RBCs) contribute to hemostasis and thrombosis by interaction with platelets via the FasL-FasR pathway to induce procoagulant activity and thrombin formation. Here, we identified a novel mechanism of platelet-RBC interaction via the CD36-thrombospondin-1 (TSP-1) signaling pathway, which is important in thrombus formation and the recruitment of RBCs to collagen-adherent platelets. Platelet-released TSP-1 can bind to CD36 at the RBC membrane to enhance procoagulant activity and to increase the activation of integrin IIb{beta}3, which represents an additional ligand for erythroid FasR, suggesting that both mechanisms of platelet-RBC interaction act in concert to propagate thrombus formation. In patients with abdominal aortic aneurysm (AAA), enhanced procoagulant activity of RBCs and platelets is accompanied by elevated exposure of TSP-1 and FasL at the platelet surface and accumulation of TSP-1 in the aortic wall and the intraluminal thrombus, suggesting that platelet-RBC interaction plays an important role in AAA pathology. TSP-1-deficient mice are protected against aortic diameter expansion in an experimental model of AAA, highlighting the crucial role of the CD36-TSP-1 axis in AAA. Thus, interfering with platelet-RBC interaction may be a promising therapeutic approach to reduce pro-coagulant activity and preserve AAA patients from surgery or rupture.

Vascular smooth muscle cell (VSMC) phenotypic switching from a contractile to synthetic state plays a pivotal role in vascular remodeling. Although genome-wide association studies have linked NINJ2 (encode Ninjurin2) polymorphisms are associated with risk of atherosclerosis and related conditions like ischemic stroke and coronary artery disease, its functional role in regulating VSMC plasticity remains unexplored. Here, using Ninj2 knockout (Ninj2-/-) and smooth muscle-specific overexpressing (Ninj2smcTG) mice subjected to carotid artery ligation, we demonstrate that Ninjurin2 deficiency exacerbates injury-induced intimal hyperplasia, whereas its overexpression attenuates this pathological remodeling. Mechanistically, Ninjurin2 knockdown promoted PDGF-BB-mediated VSMC phenotypic switching, proliferation, and migration, while biochemical studies revealed that Ninjurin2 interacts with PDGFR{beta} to suppress FAK signaling. These findings identify Ninjurin2 as a novel regulator of VSMC phenotypic switching via the PDGFR{beta}/FAK axis, offering new mechanistic insights into vascular remodeling and potential therapeutic avenues for atherosclerosis.

Chemotherapy-induced neuropathic pain (CINP) is a frequent and debilitating adverse effect of anti-tumor therapies, for which current treatments are largely non-specific and offer limited efficacy. Identifying molecular mechanisms that drive CINP may enable the development of targeted therapeutic strategies. Here, we demonstrate that paclitaxel-induced mechanical pain hypersensitivity in mice occurs independently of classical Nav1.8+ nociceptors but critically depends on TrkB+ sensory neurons. Transcriptomic analysis of TrkB+ sensory neurons revealed selective expression of Scn5a, which encodes the voltage-gated sodium channel Nav1.5, a channel classically associated with cardiac excitability. Importantly, SCN5A expression was also detected in human primary sensory neurons, indicating potential translational relevance. Functional studies further showed that Scn5a knockdown, using small interfering RNA, significantly attenuates paclitaxel-induced mechanical pain hypersensitivity. Together, these findings identify TrkB+ sensory neurons as key drivers of CINP and reveal Nav1.5 as a previously unrecognized contributor to chemotherapy-induced neuropathic pain. Targeting Nav1.5 in TrkB+ sensory neurons may therefore represent a novel therapeutic strategy for the treatment of CINP.

For patients with obesity and metabolic syndrome, bariatric procedures such as vertical sleeve gastrectomy (VSG) have a clear benefit in ameliorating metabolic dysfunction-associated steatohepatitis (MASH). While the effects of bariatric surgeries have been mainly attributed to nutrient restriction and malabsorption, whether immuno-modulatory mechanisms are involved remains unclear. Here we report that VSG ameliorates MASH progression in a weight loss- independent manner. Single-cell RNA sequencing revealed that hepatic lipid-associated macrophages (LAMs) expressing the triggering receptor expressed on myeloid cells 2 (TREM2) increase their lysosomal activity and repress inflammation in response to VSG. Remarkably, TREM2 deficiency in mice ablates the reparative effects of VSG, suggesting that TREM2 is required for MASH resolution. Mechanistically, TREM2 prevents the inflammatory activation of macrophages and is required for their efferocytotic function. Overall, our findings indicate that bariatric surgery improves MASH through a reparative process driven by hepatic LAMs, providing insights into the mechanisms of disease reversal that may result in new therapies and improved surgical interventions.

To address the concern that polygenic hazard scores for prostate cancer (PCa) might not distinguish between indolent and aggressive disease, we performed case-only analyses using a 601-variant polygenic score (PHS601). We hypothesized that among men who eventually developed PCa, those with higher PHS were more likely to develop aggressive disease. We analyzed genetic and phenotypic data from a diverse, national cohort of men diagnosed with PCa (Million Veteran Program, n = 69,901, 6413 metastatic). We used Cox proportional hazards models to examine the association of PHS601 with both age at onset of metastatic PCa (birth-to-met) and time from localized to metastatic diagnosis (localized-to-met). We performed an external validation of the case-only birth-to-met analysis in two population-based cohorts from the PRACTICAL Consortium. PHS601 was associated with both birth-to-met and localized-to-met within MVP. The HRs for men in the highest quintile of PHS601 vs. those in the lowest quintile (HR80/20) were 1.74 [1.65-1.84] and 1.41 [1.31-1.41] for birth-to-met and localized-to-met, respectively. These findings were validated in two external cohorts (COSM and ProtecT). PHS601 was also associated with earlier development of metastasis. Men with high PHS601 are diagnosed with prostate cancer at a younger age and are more likely to develop metastasis.

Neutrophil extracellular traps (NETs) drive atherosclerosis progression and are associated with adverse clinical outcomes like myocardial infarction and stroke. While the triggers of NETosis in atherosclerotic plaque are well-characterized, the mechanisms underlying NET degradation and clearance remain unclear. Moreover, the impact of impaired NET clearance on atherosclerosis progression have not been elucidated. Here we show that macrophages are critical for the release of DNases, which degrade NETs. We identified endoplasmic reticulum (ER) stress mediated activation of PERK-ATF4 pathway as a key driver of impaired macrophage DNase secretion, leading to delayed NET clearance and their persistence. Elevated NET levels trigger cleavage of the efferocytosis receptor Mertk leading to defective macrophage efferocytosis and exacerbation of plaque necrosis. Human atherosclerotic plaques and Ldlr-/- mice treated with ISRIB, a PERK inhibitor, show enhanced DNase secretion and clearance of NETs. Together, the identification of key mechanisms of NET clearance in atherosclerosis offers new therapeutic strategies to stabilize plaques.

SARS-CoV-2 precipitates respiratory distress by infection of airway epithelial cells and is often accompanied by acute kidney injury. We report that Kidney Injury Molecule-1/T cell immunoglobulin mucin domain 1 (KIM-1/TIM-1) is expressed in lung and kidney epithelial cells in COVID-19 patients and is a receptor for SARS-CoV-2. Human and mouse lung and kidney epithelial cells express KIM-1 and endocytose nanoparticles displaying the SARS-CoV-2 spike protein (virosomes). Uptake was inhibited by anti-KIM-1 antibodies and TW-37, a newly discovered inhibitor of KIM-1-mediated endocytosis. Enhanced KIM-1 expression by human kidney tubuloids increased uptake of virosomes. KIM-1 binds to the SARS-CoV-2 Spike protein in vitro. KIM-1 expressing cells, not expressing angiotensin-converting enzyme 2 (ACE2), are permissive to SARS-CoV-2 infection. Thus, KIM-1 is an alternative receptor to ACE2 for SARS-CoV-2. KIM-1 targeted therapeutics may prevent and/or treat COVID-19.

Aggressive variant prostate cancer (AVPC) is a lethal subtype of prostate cancer characterized by its androgen independence, resistance to chemotherapy, and display of neuroendocrine features which can emerge either de novo or via transformation after a prior diagnosis of adenocarcinoma. The poor clinical outcomes in patients with AVPC are associated with its profound molecular heterogeneity. In this study, we analyzed 23 consecutive AVPC cases treated at a dedicated small-cell clinic (2017-2025) using clinicogenomic and transcriptomic profiling. Transformed AVPC exhibited significantly shorter overall survival times than de novo AVPC (11.8 vs 26.0 months, P < 0.001). Integrative genomic analyses identified residual androgen signaling in subsets of cases harboring neuroendocrine lineage programs, highlighting a decoupling of lineage identity and morphology. To facilitate mechanistic and pharmacologic studies, we established NCI-LYM-1, a patient-derived organoid/PDX from an AR-negative, ASCL1+/SYP+ lymph node metastasis, which faithfully recapitulates the donor tumors molecular and phenotypic features. Short- and long-read whole-genome sequencing combined with optical genome mapping identified biallelic inactivation of PTEN, TP53, RB1 and BRCA2 as potential drivers, demonstrating clonal concordance with circulating tumor DNA from the original patient donor. Pathway and perturbation analyses suggested that NCI-LYM-1 harbored a strong dependency on apoptotic pathways, which was confirmed by in vitro organoid testing with the BCL-2/BCL-xL inhibitor navitoclax (IC50: 0.27 {micro}M) and the MCL-1 inhibitor AZD-5991 (IC50: 0.060 {micro}M). Overall, NCI-LYM-1 recapitulates the clinical aggressiveness and heterogeneity of AVPC, providing a tractable platform to identify novel precision therapies.

Non-ICANS neurotoxicities (NINTs) are serious, atypical toxicities associated with ciltacabtagene autoleucel, a commercial chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory multiple myeloma. Risk factors contributing to the development of NINTs are poorly understood. In a cohort of 109 patients, we identify predisposing risk factors and propose strategies to mitigate NINTs. We show that high peak absolute lymphocyte count is a strong NINT predictor which directly correlates with flow cytometry-based peripheral blood CAR T cell quantitation. The observed CAR lymphocytosis was polyclonal with a bias towards CD4+ CAR T cells rich in memory marker expression. We then identified CAR lymphocytosis associated CD4+ CAR T cell populations which exhibited increased inflammatory pathway gene expression. Finally, we characterize NINT associated CD4+ CAR T cell populations which are potential therapeutic targets for future exploration. One Sentence SummaryCiltacabtagene autoleucel associated non-ICANS neurotoxicities are driven by high CD4+ CAR T cell expansion exhibiting memory marker expression and upregulated inflammatory gene signaling pathways.