Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission.

Despite a high response rate in chimeric antigen receptor (CAR) T cell therapy for acute lymphocytic leukaemia (ALL)1-3, approximately 50% of patients relapse within the first year4-6, representing an urgent question to address in the next stage of cellular immunotherapy. Here, to investigate the molecular determinants of ultralong CAR T cell persistence, we obtained a single-cell multi-omics atlas from 695,819 pre-infusion CAR T cells at the basal level or after CAR-specific stimulation from 82 paediatric patients with ALL enrolled in the first two CAR T ALL clinical trials and 6 healthy donors. We identified that elevated type 2 functionality in CAR T infusion products is significantly associated with patients maintaining a median B cell aplasia duration of 8.4 years. Analysis of ligand-receptor interactions revealed that type 2 cells regulate a dysfunctional subset to maintain whole-population homeostasis, and the addition of IL-4 during antigen-specific activation alleviates CAR T cell dysfunction while enhancing fitness at both transcriptomic and epigenomic levels. Serial proteomic profiling of sera after treatment revealed a higher level of circulating type 2 cytokines in 5-year or 8-year relapse-free responders. In a leukaemic mouse model, type 2high CAR T cell products demonstrated superior expansion and antitumour activity, particularly after leukaemia rechallenge. Restoring antitumour efficacy in type 2low CAR T cells was attainable by enhancing their type 2 functionality, either through incorporating IL-4 into the manufacturing process or by priming manufactured CAR T products with IL-4 before infusion. Our findings provide insights into the mediators of durable CAR T therapy response and suggest potential therapeutic strategies to sustain long-term remission by boosting type 2 functionality in CAR T cells.

A systematic review of immunotherapy in high-grade glioma: learning from the past to shape future perspectives.

High-grade gliomas (HGGs) constitute the most common malignant primary brain tumor with a poor prognosis despite the standard multimodal therapy. In recent years, immunotherapy has changed the prognosis of many cancers, increasing the hope for HGG therapy. We conducted a comprehensive search on PubMed, Scopus, Embase, and Web of Science databases to include relevant studies. This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Fifty-two papers were finally included (44 phase II and eight phase III clinical trials) and further divided into four different subgroups: 14 peptide vaccine trials, 15 dendritic cell vaccination (DCV) trials, six immune checkpoint inhibitor (ICI) trials, and 17 miscellaneous group trials that included both "active" and "passive" immunotherapies. In the last decade, immunotherapy created great hope to increase the survival of patients affected by HGGs; however, it has yielded mostly dismal results in the setting of phase III clinical trials. An in-depth analysis of these clinical results provides clues about common patterns that have led to failures at the clinical level and helps shape the perspective for the next generation of immunotherapies in neuro-oncology.

CSF-based liquid biopsy pointing to a diagnosis of diffuse glioma in a patient with supposed neurodegenerative disorder.

INTRODUCTION: The differential diagnosis of brain diseases becomes challenging in cases where imaging is not sufficiently informative, and surgical biopsy is impossible or unacceptable to the patient. METHODS: An elderly patient with progressive short-term memory loss and cognitive impairment presented with a normal brain CT scan, a brain FDG-PET that indicated symmetrical deterioration of the white matter in the frontal lobes, and inconclusive results of a molecular marker analysis of suspected dementia in cerebrospinal fluid (CSF). Brain MRI suggested the diagnosis of lower grade glioma. The patient refused surgical biopsy. In order to investigate whether somatic mutations associated with gliomas existed, we performed a "liquid biopsy" by the targeted sequencing of cell-free DNA (cfDNA) from his CSF. RESULTS: Deep sequencing of the cfDNA from CSF revealed somatic mutations characteristically found in gliomas, including mutations of the TP53 (Arg282Trp), BRAF (Val600Glu), and IDH1 (Arg132His) genes. The patient is currently treated with temozolomide, and his clinical and MRI findings suggest the stabilization of his disease. CONCLUSION: Neurological patients may benefit from liquid biopsy diagnostic work-up as it can reveal therapeutically targetable mutations.

Reassessing the efficacy of bevacizumab in newly diagnosed glioblastoma: A systematic review and external pseudodata-based analysis.

Background: First-line use of bevacizumab for glioblastoma (GBM) was evaluated in 2 phase 3 randomized controlled trials (RCT), demonstrating an impact on progression-free survival but not overall survival (OS). However, the crossover events of these trials raised concerns regarding the reliability of this latter analysis. In this study, we conducted an external control-based reassessment of the bevacizumab efficacy in newly diagnosed GBM (ndGBM) against the standard Stupp protocol. Methods: A systematic review of the literature was conducted to identify the phase 3 RCTs in ndGBM incorporating the Stupp protocol as an arm. For the selected studies, we extracted individual patient survival pseudodata of the Stupp protocol arm by digitizing the Kaplan-Meier plots. A comprehensive pipeline was established to select suitable control studies as external benchmarks. Results: Among the 13 identified studies identified in our systematic review, 4 studies resulted as comparable with the AVAglio trial and 2 with the RTOG 0825. Pooled individual patient pseudodata analysis showed no differences in terms of OS when bevacizumab was added to the Stupp protocol. Conclusions: The external-controlled-based reassessment of the bevacizumab treatment in ndGBM confirmed its lack of efficacy in extending OS. Our study includes a summary table of individual patient survival pseudodata from all phase 3 RCTs in ndGBM employing the Stupp protocol and provides a pipeline that offers comprehensive guidance for conducting external control-based assessments in ndGBM.

CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors.

In the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach for combating cancers, demonstrating remarkable efficacy in relapsed/refractory hematological malignancies in both pediatric and adult patients. CAR-natural killer (CAR-NK) cell complements CAR-T cell therapy by offering several distinct advantages. CAR-NK cells do not require HLA compatibility and exhibit low safety concerns. Moreover, CAR-NK cells are conducive to "off-the-shelf" therapeutics, providing significant logistic advantages over CAR-T cells. Both CAR-T and CAR-NK cells have shown consistent and promising results in hematological malignancies. However, their efficacy against solid tumors remains limited due to various obstacles including limited tumor trafficking and infiltration, as well as an immuno-suppressive tumor microenvironment. In this review, we discuss the recent advances and current challenges of CAR-T and CAR-NK cell immunotherapies, with a specific focus on the obstacles to their application in solid tumors. We also analyze in depth the advantages and drawbacks of CAR-NK cells compared to CAR-T cells and highlight CAR-NK CAR optimization. Finally, we explore future perspectives of these adoptive immunotherapies, highlighting the increasing contribution of cutting-edge biotechnological tools in shaping the next generation of cellular immunotherapy.

Unveiling the SOD1-mediated ALS phenotype: insights from a comprehensive meta-analysis.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis associated with mutations in SOD1 (SOD1-ALS) might be susceptible to specific treatment. The aim of the study is to outline the clinical features of SOD1-ALS patients by comparing them to patients without ALS major gene variants and patients with variants in other major ALS genes. Defining SOD1-ALS phenotype may assist clinicians in identifying patients who should be prioritized for genetic testing. METHODS: We performed an extensive literature research including original studies which reported the clinical features of SOD1-ALS and at least one of the following patient groups: C9ORF72 hexanucleotide repeat expansion (C9-ALS), TARDBP (TARDBP-ALS), FUS (FUS-ALS) or patients without a positive test for a major-ALS gene (N-ALS). A random effects meta-analytic model was applied to clinical data extracted encompassing sex, site and age of onset. To reconstruct individual patient survival data, the published Kaplan-Meier curves were digitized. Data were measured as odds ratio (OR) or standardized mean difference (SMD) as appropriate. Median survival was compared between groups. RESULTS: Twenty studies met the inclusion criteria. We identified 721 SOD1-ALS, 470 C9-ALS, 183 TARDBP-ALS, 113 FUS-ALS and 2824 N-ALS. SOD1-ALS showed a higher rate of spinal onset compared with N-ALS and C9-ALS (OR = 4.85, 95% CI = 3.04-7.76; OR = 10.47, 95% CI = 4.32-27.87) and an earlier onset compared with N-ALS (SMD = - 0.45, 95% CI = - 0.72 to - 0.18). SOD1-ALS had a similar survival compared with N-ALS (p = 0.14), a longer survival compared with C9-ALS (p < 0.01) and FUS-ALS (p = 0.019) and a shorter survival compared with TARDBP-ALS (p < 0.01). DISCUSSION: This study indicates the presence of a specific SOD1-ALS phenotype. Insights in SOD1-ALS clinical features are important in genetic counseling, disease prognosis and support patients' stratification in clinical trials.

Caudate functional networks influence brain structural changes with aging.

Neurogenesis decline with aging may be associated with brain atrophy. Subventricular zone neuron precursor cells possibly modulate striatal neuronal activity via the release of soluble molecules. Neurogenesis decay in the subventricular zone may result in structural alterations of brain regions connected to the caudate, particularly to its medial component. The aim of this study was to investigate how the functional organization of caudate networks relates to structural brain changes with aging. One hundred and fifty-two normal subjects were recruited: 52 young healthy adults (≤35 years old), 42 middle-aged (36 ≤ 60 years old) and 58 elderly subjects (≥60 years old). In young adults, stepwise functional connectivity was used to characterize regions that connect to the medial and lateral caudate at different levels of link-step distances. A statistical comparison between the connectivity of medial and lateral caudate in young subjects was useful to define medial and lateral caudate connected regions. Atrophy of medial and lateral caudate connected regions was estimated in young, middle-aged and elderly subjects using T1-weighted images. Results showed that middle-aged and elderly adults exhibited decreased stepwise functional connectivity at one-link step from the caudate, particularly in the frontal, parietal, temporal and occipital brain regions, compared to young subjects. Elderly individuals showed increased stepwise functional connectivity in frontal, parietal, temporal and occipital lobes compared to both young and middle-aged adults. Additionally, elderly adults displayed decreased stepwise functional connectivity compared to middle-aged subjects in specific parietal and subcortical areas. Moreover, in young adults, the medial caudate showed higher direct connectivity to the basal ganglia (left thalamus), superior, middle and inferior frontal and inferior parietal gyri (medial caudate connected region) relative to the lateral caudate. Considering the opposite contrast, lateral caudate showed stronger connectivity to the basal ganglia (right pallidum), orbitofrontal, rostral anterior cingulate and insula cortices (lateral caudate connected region) compared to medial caudate. In elderly subjects, the medial caudate connected region showed greater atrophy relative to the lateral caudate connected region. Brain regions linked to the medial caudate appear to be more vulnerable to aging than lateral caudate connected areas. The adjacency to the subventricular zone may, at least partially, explain these findings. Stepwise functional connectivity analysis can be useful to evaluate the role of the subventricular zone in network disruptions in age-related neurodegenerative disorders.

In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy.

Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

Muscle hypertrophy following acquired neurogenic injury: systematic review and analysis of existing literature.

OBJECTIVES: Neurogenic muscle hypertrophy (NMH) is a rare condition characterized by focal muscle hypertrophy caused by chronic partial nervous injury. Given its infrequency, underlying mechanisms remain poorly understood. Inspired by two clinical cases, we conducted a systematic review to gain insights into the different aspects of NMH. METHODS: We systematically searched online databases up until May 30, 2023, for reports of muscle hypertrophy attributed to acquired neurogenic factors. We conducted an exploratory analysis to identify commonly associated features. We also report two representative clinical cases. RESULTS: Our search identified 63 reports, describing 93 NMH cases, to which we added our two cases. NMH predominantly affects patients with compressive radiculopathy (68.4%), negligible muscular weakness (93.3%), and a chronic increase in muscle bulk. A striking finding in most neurophysiological studies (60.0%) is profuse spontaneous discharges, often hindering the analysis of voluntary traces. Some patients exhibited features consistent with more significant muscle damage, including higher creatine phosphokinase levels, muscle pain, and inflammatory muscle infiltration. These patients are sometimes referred to in literature as "focal myositis." Treatment encompassed corticosteroid, Botulinum Toxin A, decompressive surgery, antiepileptic medications, and nerve blocks, demonstrating varying degrees of efficacy. Botulinum Toxin A yielded the most favorable response in terms of reducing spontaneous discharges. INTERPRETATION: This systematic review aims to provide a clear description and categorization of this uncommon presentation of an often-overlooked neurological disorder. Though questions remain about the underlying mechanism, evidence suggests that aberrant fiber overstimulation along with increased workload that promotes focal damage may result in muscle hypertrophy. This may serve as a guide for therapeutic interventions.

Evaluation of the combined effect of mobility and seasonality on the COVID-19 pandemic: a Lombardy-based study.

Restrictions to human mobility had a significant role in limiting SARS-CoV-2 spread. It has been suggested that seasonality might affect viral transmissibility. Our study retrospectively investigates the combined effect that seasonal environmental factors and human mobility played on transmissibility of SARS-CoV-2 in Lombardy, Italy, in 2020. Environmental data were collected from accredited open-source web services. Aggregated mobility data for different points of interests were collected from Google Community Reports. The Reproduction number (Rt), based on the weekly counts of confirmed symptomatic COVID-19, non-imported cases, was used as a proxy for SARS-CoV-2 transmissibility. Assuming a non-linear correlation between selected variables, we used a Generalized Additive Model (GAM) to investigate with univariate and multivariate analyses the association between seasonal environmental factors (UV-index, temperature, humidity, and atmospheric pressure), location-specific mobility indices, and Rt. UV-index was the most effective environmental variable in predicting Rt. An optimal two-week lag-effect between changes in explanatory variables and Rt was selected. The association between Rt variations and individually taken mobility indices differed: Grocery & Pharmacy, Transit Station and Workplaces displayed the best performances in predicting Rt when individually added to the multivariate model together with UV-index, accounting for 85.0%, 85.5% and 82.6% of Rt variance, respectively. According to our results, both seasonality and social interaction policies played a significant role in curbing the pandemic. Non-linear models including UV-index and location-specific mobility indices can predict a considerable amount of SARS-CoV-2 transmissibility in Lombardy during 2020, emphasizing the importance of social distancing policies to keep viral transmissibility under control, especially during colder months.

Transcriptional effects of fingolimod treatment on peripheral T cells in relapsing remitting multiple sclerosis patients.

Aim: To investigate the transcriptional changes induced by Fingolimod (FTY) in T cells of relapsing remitting multiple sclerosis patients. Patients & methods: Transcriptomic changes after 6 months of FTY therapy were evaluated on T cells from 24 relapsing remitting multiple sclerosis patients through RNA-sequencing, followed by technical validation and pathway analysis. Results: Among differentially expressed genes, CX3CR1 and CCR7 resulted strongly up- and downregulated, respectively. Two relevant genes were validated with quantitative PCR and we largely confirmed findings from two previous microarray-based studies with similar design. Pathway analysis pointed to an involvement of processes related to immune function and cell migration. Conclusion: Our data support the evidence that FTY induces major transcriptional changes in genes involved in immune response and cell trafficking in T lymphocytes.

Neurofilament light chain as a biological marker for amyotrophic lateral sclerosis: a meta-analysis study.

Aim: The aim of the present metanalysis is to evaluate blood and CSF Neurofilament light chain (NfL) concentrations in ALS patients, compared to healthy controls, ALS mimic disorders (ALSmd) and other neurological diseases (OND), and to evaluate their diagnostic yield against ALSmd. Methods: Search engines were systematically investigated for relevant studies. A random effect model was applied to estimate the pooled standard mean difference in NfL levels between ALS and controls and a bivariate mixed-effects model was applied to estimate their diagnostic accuracy on blood and CSF. Results and conclusions: NfL CSF levels were higher in ALS compared with all other control groups. On blood, NfL levels were significantly higher in ALS patients compared with healthy controls and ALSmd. In a subgroup analysis, the use of SIMOA yielded to a better differentiation between ALS and controls on blood, compared with ELISA. Studies performed on CSF (AUC = 0.90) yielded to better diagnostic performances compared with those conducted on blood (AUC = 0.78). Further prospective investigations are needed to determine a diagnostic cutoff, exploitable in clinical practice.

Neural precursor cells tune striatal connectivity through the release of IGFBPL1.

The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.

Endogenous neural precursor cells in health and disease.

Neurogenesis persists in the adult brain of mammals in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus (DG). The complex interactions between intrinsic and extrinsic signals provided by cells in the niche but also from distant sources regulate the fate of neural stem/progenitor cells (NPCs) in these sites. This fine regulation is perturbed in aging and in pathological conditions leading to a different NPC behavior, tailored to the specific physio-pathological features. Indeed, NPCs exert in physiological and pathological conditions important neurogenic and non-neurogenic regulatory functions and participate in maintaining and protecting brain tissue homeostasis. In this review, we discuss intrinsic and extrinsic signals that regulate NPC activation and NPC functional role in various homeostatic and non-homeostatic conditions.