Chimeric antigen receptor (CAR) T cells have shown remarkable therapeutic promise in hematological malignancies and, more recently, in autoimmunity. They also hold considerable potential for neurodegenerative diseases and CNS injury, where the therapeutic objective shifts from cell depletion to immune modulation and tissue repair. Using ischemic stroke as proof-of-concept, we engineered MOG-targeting CAR T cells to dissect how distinct CAR designs shape the CNS microenvironment. CD4/CD8 CAR T cells (a mixture of CD4⁺ and CD8⁺ subsets) proliferated robustly and efficiently infiltrated the ischemic hemisphere, but induced broad immune recruitment and exacerbated neuroinflammation. In contrast, CD4⁺ restricted CAR T cells markedly reduced immune infiltration, reprogrammed microglia, and minimized inflammatory activation. We engineered CD4⁺ CAR T cells to secrete brain-derived neurotrophic factor (BDNF) to determine whether they could be redirected toward a reparative, non-cytotoxic phenotype. CD4⁺ BDNF-CAR T cells further attenuated inflammation, reduced immune infiltration, and promoted the expansion of regulatory T cells. CD4⁺ BDNF-CAR T treated mice showed significantly improved gait performance following stroke. Together, these findings establish a cellular framework and outline principles for engineering reparative CAR T platforms for neurological diseases. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 101055341, 101095540, 101123436 Deutsche Forschungsgemeinschaft, 259373024 Israel Science Foundation, 1944/22 United States-Israel Binational Science Foundation, https://ror.org/00j8z2m73 Ministry of Innovation, Science and Technology, 1001703362 Alzheimer’s Association, ABA-25-1373817

Abstract Despite major therapeutic advances, a substantial fraction of patients with autoimmune disease remains refractory to treatment. While B cell-targeted CAR-T therapies have shown considerable efficacy, the central contribution of pathogenic T cells to rheumatoid arthritis (RA) suggests that complementary T cell-directed strategies may enable deeper disease control. Using single-cell multi-omics of human RA and experimental models, PDCD1 was identified as a selective marker of synovial disease-associated T cells. We developed PD-1-directed CAR-T cells that potently eliminate these cells in vitro and in vivo , leading to marked attenuation of synovitis in RA models. To limit off-target activity, we engineered NR4A2-driven CAR-responsive biosensors to restrict CAR activity to inflamed synovium. To couple anti-PD-1 CAR-mediated cytotoxicity with microenvironmental modulation, we further engineered these CAR-T cells to secrete soluble TNF receptor II (sTNFRii), counteracting baseline inflammation and CAR-induced IFN response and promoting a tissue-reparative myeloid state. PD-1-targeted CAR-T therapy thus represents a promising, specific, and safe strategy for autoimmune diseases involving disease-associated T cells.

B cell-depleting therapies are effective in multiple sclerosis (MS), yet some patients relapse, underscoring the need for more precise interventions. To identify new therapeutic targets, we generated a single-cell RNA sequencing (scRNA-seq) atlas of cerebrospinal fluid (CSF), brain, and blood from non-inflammatory controls and patients with MS or other neuroinflammatory diseases. We found disease-associated enrichment of class-switched immunoglobulin G+ (IgG+) B cells and plasma cells in MS CSF. Unbiased analysis identified a rare disease-enriched subset of activated, T cell receptor (TCR)-restricted, PD-1+ T follicular helper-like cells with B cell-recruiting features. To target this population, we developed PD-1-directed chimeric antigen receptor (CAR) T cells that selectively depleted pathogenic PD-1+ CD4 T cells and locally released IL-10. This strategy attenuated central nervous system (CNS) inflammation, reprogrammed the local immune milieu, and improved clinical outcomes across murine neuroinflammation models. These findings define a CNS-localized adaptive immune circuit in MS and nominate programmable PD-1 CAR T cells as a strategy to disrupt it.

Non-canonical metabolic functions of signaling molecules contribute to cancer plasticity and metastatic progression. Here, we demonstrated that PDE5a inhibitors, including sildenafil (Viagra), induced lysosomal cholesterol accumulation across multiple mouse and human cancer models, reducing cholesterol bioavailability and impairing cancer cell migration and metastasis. Cancer cells exhibited heightened sensitivity due to reduced lysosomal gene expression, rendering them particularly vulnerable to disrupted cholesterol trafficking. Mechanistically, elevated cGMP bound the lysosomal cholesterol transporter NPC1, impairing cholesterol export and phenocopying Niemann-Pick type C pathology. The resulting cholesterol depletion disrupted membrane lipid rafts and mitochondrial bioenergetics, thereby limiting metastatic capacity and triggering compensatory SREBP2 activation with increased cholesterol synthesis. Combining sildenafil with statins yields additive antimetastatic effects by concurrently blocking lysosomal cholesterol export and cholesterol biosynthesis. Consistently, analysis of digital health records demonstrated significantly improved survival among sildenafil users, with a dose-dependent additive benefit observed when combined with statins. Together, these findings identify increasing cGMP levels through PDE5a inhibition as a potential strategy to restrict metastasis and offer a potential mechanistic basis for the beneficial effects of sildenafil.

Supplementary Figure S2 demonstrates the infiltration of mature innate immune cells to the liver during breast carcinogenesis

Supplementary Table S4 lists the antibodies used for FACS

Supplementary Extended Data 1 describes the FACS gating startegy

Supplementary Figure S1 demonstrates global metabolic changes in liver metabolism during early BC carcinogenesis

Argininosuccinate synthetase 1 (ASS1) expression and arginine availability are key metabolic determinants that influence tumor fitness and regulate immune interactions within the tumor microenvironment (TME). Using an orthotopic triple-negative breast cancer (TNBC) model, we demonstrate that arginine deprivation heightens tumor dependence on the TME for survival. Mechanistically, fibroblasts sustain tumor viability by supplying arginine, whereas macrophages cooperate with stromal cues to activate Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling, thereby enhancing tumor survival. Concordantly, a JAK-STAT gene-expression signature correlates with ASS1 levels in human TNBC datasets. Translationally, combined pharmacological inhibition of JAK signaling with arginine deprivation markedly suppresses tumor growth. Together, these findings reveal a TME-driven, targetable stromal–immune circuit that enables tumors to withstand arginine deficiency-induced metabolic stress. Broadly, our work highlights that mapping and strategically inducing metabolic dependencies can reveal actionable compensatory pathways, offering opportunities to improve cancer therapy.

Supplementary Figure S4 demonstrates similar transcriptional and metabolic changes following liver infiltration of immune cells to the liver of PDAC mice

Supplementary Table S1 details clinical data of PDAC patients

Supplementary Figure S3 shows that targeting the IL-6-pSTAT-HNF4a pathway preserves liver metabolism and restricts BC growth

Supplementary Figure S5 demonstrate the significance of our liver score in predicting outcomes of BC and PDAC patients

Abstract Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6–pSTAT3 immune–hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients’ outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. Significance: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501

Intravital imaging of brain vasculature through the intact cranium in vivo is based on the evolution of the fluorescence intensity and provides an ability to characterize various physiological processes in the natural context of cellular resolution. The involuntary motions of the examined subjects often limit in vivo non-invasive functional optical imaging. Conventional imaging diagnostic modalities encounter serious difficulties in correction of artificial motions, associated with fast high dynamics of the intensity values in the collected image sequences, when a common reference cannot be provided. In the current report, we introduce an alternative solution based on a time-space Fourier transform method so-called K-Omega. We demonstrate that the proposed approach is effective for image stabilization of fast dynamic image sequences and can be used autonomously without supervision and assignation of a reference image.

Argininosuccinate synthase (ASS1) downregulation in different tumors has been shown to support cell proliferation and yet, in several common cancer subsets ASS1 expression associates with poor patient prognosis. Here we demonstrate that ASS1 expression under glucose deprivation is induced by c-MYC, providing survival benefit by increasing nitric oxide synthesis and activating the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase by S-nitrosylation. The resulting increased flux through gluconeogenesis enhances serine, glycine and subsequently purine synthesis. Notably, high ASS1-expressing breast cancer mice do not respond to immune checkpoint inhibitors and patients with breast cancer with high ASS1 have more metastases. We further find that inhibiting purine synthesis increases pyrimidine to purine ratio, elevates expression of the immunoproteasome and significantly enhances the response of autologous primary CD8 + T cells to anti-PD-1. These results suggest that treating patients with high-ASS1 cancers with purine synthesis inhibition is beneficial and may also sensitize them to immune checkpoint inhibition therapy.

Functional handwriting is a process involving various complex interactions between physical, cognitive and sensory systems. Since muscular motion is of a peculiar nature for each person, handwriting properties, such as pencil pressure and speed of writing, can be considered as a unique marker of identity. Moreover, impairments of handwriting in many cases are connected to neurodevelopmental disorders such as attention deficit hyperactivity disorder, developmental coordination disorder, autism spectrum disorders, Parkinson's disease, etc. From this point of view, investigations of handwriting kinematics and pressure can be highly important for both forensic science and medicine. Commonly, the kinematic and pressure features of handwriting are evaluated using a graphics tablet with a stylus or electronic pens. The production of such devices is quite expensive. Therefore, the development of new methods for individual handwriting analysis is an important and current goal. Laser speckle contrast imaging (LSCI) is a powerful method, which is sensitive to both motion and pressure. Since the developed technique requires the use of only a simple laser diode and camera for image acquisition, LSCI is a cost-effective and practical tool for handwriting analysis. In the current letter we present a robust LSCI-based method for handwriting pressure and kinematics evaluation. The introduced approach was validated by an Archimedean spiral writing task.

Patients with germline mutations in the urea-cycle enzyme argininosuccinate lyase (ASL) are at risk for developing neurobehavioral and cognitive deficits. We find that ASL is prominently expressed in the nucleus locus coeruleus (LC), the central source of norepinephrine. Using natural history data, we show that individuals with ASL deficiency are at risk for developing attention deficits. By generating LC-ASL-conditional knockout (cKO) mice, we further demonstrate altered response to stressful stimuli with increased seizure reactivity in LC-ASL-cKO mice. Depletion of ASL in LC neurons leads to reduced amount and activity of tyrosine hydroxylase (TH) and to decreased catecholamines synthesis, due to decreased nitric oxide (NO) signaling. NO donors normalize catecholamine levels in the LC, seizure sensitivity, and the stress response in LC-ASL-cKO mice. Our data emphasize ASL importance for the metabolic regulation of LC function with translational relevance for ASL deficiency (ASLD) patients as well as for LC-related pathologies.

Laser speckle imaging (LSI) is a well-known and useful approach for the non-invasive visualization of flows and microcirculation localized in turbid scattering media, including biological tissues (such as brain vasculature, skin capillaries etc.). Despite an extensive use of LSI for brain imaging, the LSI technique has several critical limitations. One of them is associated with inability to resolve a functionality of vessels. This limitation also leads to the systematic error in the quantitative interpretation of values of speckle contrast obtained for different vessel types, such as sagittal sinus, arteries, and veins. Here, utilizing a combined use of LSI and fluorescent intravital microscopy (FIM), we present a simple and robust method to overcome the limitations mentioned above for the LSI approach. The proposed technique provides more relevant, abundant, and valuable information regarding perfusion rate ration between different types of vessels that makes this method highly useful for in vivo brain surgical operations.

Optical clearing agents (OCAs) and many chemicals are widely used in functional diagnosis of skin tissues. Numerous studies are associated with the transcutaneous diffusion of OCA in epidermal, dermal, and hypodermal tissues, which results in changing their optical properties. In addition, an objective approach that is suitable for screening the influence of utilized OCA, as well as various chemical agents, synthetics, and nanomaterials, on blood and lymph flows is highly desirable. In our study, a highly sensitive laser speckle imaging (LSI) system and fluorescent intravital microscopy (FIM) were used team-wise to inspect the acute skin vascular permeability reaction in mouse ear during the local application of OCA on the skin surface. Fluorescent contrast material administrated intravenously was used for quantitatively assessing the intensity of vascular permeability reaction and the strength of skin irritation. The obtained results suggest that a combined use of LSI and FIM is highly effective for monitoring the cutaneous vascular permeability reaction, with great potential for assessment of allergic reactions of skin in response to interactions with chemical substances.