Whole organism snRNA-seq reveals systemic peripheral changes in Alzheimer's Disease fly models.

Peripheral tissues become disrupted in Alzheimer's Disease (AD). However, a comprehensive understanding of how the expression of AD-associated toxic proteins, Aß42 and Tau, in neurons impacts the periphery is lacking. Using Drosophila, a prime model organism for studying aging and neurodegeneration, we generated the Alzheimer's Disease Fly Cell Atlas (AD-FCA): whole-organism single-nucleus transcriptomes of 219 cell types from adult flies neuronally expressing human Aß42 or Tau. In-depth analyses and functional data reveal impacts on peripheral sensory neurons by Aß42 and on various non-neuronal peripheral tissues by Tau, including the gut, fat body, and reproductive system. This novel AD atlas provides valuable insights into potential biomarkers and the intricate interplay between the nervous system and peripheral tissues in response to AD-associated proteins.

Anti-pancreatic cancer activity of cassane diterpenoids isolated from the seeds of Caesalpinia sappan mediated by autophagy activation via ROS/AMPK/mTORC1 pathway.

Three undescribed cassane diterpenoids, caesalpanins D-F (1-3), and seven known ones were isolated from the seeds of Caesalpinia sappan. Structures and absolute configurations of 1-3 were elucidated based on the extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, and ECD calculations. Structurally, compound 1 was the first example of 18-norcassane diterpenoid and 2 was a rare 20-norcassane diterpenoid having an unusual five-membered oxygen bridge between C-10/C-18. The anti-proliferative activity of 1, 3, and 4-10 against PANC-1 cells (pancreatic ductal adenocarcinoma cell line) was evaluated, and phanginin H (4) was found to exhibit anti-cancer activity with IC50 value of 18.13 ± 0.63 µM. Compound 4 inhibited PANC-1 cell growth by arresting the cell cycle at G2/M phase via regulation of cyclin-dependent kinases, and the self-renewal and metastasis of PANC-1 cells by suppressing cancer cell stemness. Furthermore, compound 4 induced ROS generation and subsequently activated autophagy, which was demonstrated by the formation of autophagic vacuoles and dynamic change of autophagic flux. The induced ROS accumulation resulted in AMPK activation and subsequently regulation of mTORC1 activity and ULK phosphorylation, indicating that 4 triggered autophagy through ROS/AMPK/mTORC1 pathway. These findings suggested that 4 might potentially be an autophagy inducer for the therapy of pancreatic cancer.

LncRNA ZFAS1 Alleviated NLRP3 Inflammasome-Mediated Pyroptosis through Regulating miR-96-5p/Smad7 Signaling in Allergic Rhinitis.

INTRODUCTION: The NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptosis was positively correlated with the allergic rhinitis progression and was reported to be regulated by SMAD family member 7 (Smad7). Bioinformatics analysis revealed that Smad7 might be targeted by miR-96-5p, and miR-96-5p might be targeted by long noncoding RNA zinc finger antisense 1 (ZFAS1). However, the effects and regulatory mechanisms of the ZFAS1/miR-96-5p/Smad7 functional axis in allergic rhinitis have not been investigated. METHODS: Human nasal mucosa epithelial cell line RPMI 2650 and C57BL/6 mice were obtained for in vitro and in vivo studies. Dual-luciferase reporter assay and RNA immunoprecipitation were implemented for detecting molecular interactions. Cell counting kit-8 and flow cytometry were used for measuring cell viability and pyroptosis. ELISA was obtained for monitoring cytokine secretion. RT-qPCR and Western blot were examined for determining RNA and protein expression. RESULTS: In vitro studies revealed that ZFAS1 was downregulated in interleukin (IL)-13-treated RPMI 2650 cells, while overexpression of ZFAS1 enhanced cell viability and inhibited NLRP3-mediated pyroptosis and inflammatory response. ZFAS1 directly inhibited miR-96-5p to suppress NLRP3-mediated pyroptosis in IL-13-treated RPMI 2650 cells. MiR-96-5p bound to the 3'-untranslated region of Smad7 and knockdown of Smad7 significantly reversed the effects of miR-96-5p depletion. Moreover, in vivo experiments further confirmed the findings of in vitro studies and showed ZFAS1 overexpression or miR-96-5p inhibition alleviated allergic rhinitis in vivo. CONCLUSION: ZFAS1 downregulated the expression of miR-96-5p to upregulate Smad7 level, which subsequently inhibited NLRP3-mediated pyroptosis and inflammatory response to ameliorate allergic rhinitis.

Prevalence and influencing factors of muscle mass loss in adults with diabetes and a high body fat percentage: A cross-sectional study.

BACKGROUND: In adults with type 2 diabetes (T2DM), sarcopenia and obesity are two common body composition issues. OBJECTIVE: We investigated the associated influencing factors of muscle mass loss in obese adults with T2DM, to provide a theoretical basis for the prevention of sarcopenic obesity in patients with T2DM. METHODS: We recruited 315 participants in this study. The participants underwent body composition assessment and clinical information was collected. Dual-energy X-ray absorptiometry was used to verify the accuracy of the body composition data. Based on their body fat percentage, 189 patients with T2DM were classified as obese. Patients with T2DM and obesity were grouped into the muscle mass loss group and non-muscle mass loss group based on gender. We collected demographic and clinical information about patients with T2DM who were obese, including their age, gender, body mass index (BMI), appendicular skeletal muscle index (ASMI), and body fat percentage (PBF). RESULTS: Among the participants who were obese and had T2DM, 56.61% (107/189) experienced muscle mass loss, with a detection rate of 43.42% (33/76) among females and 65.49% (74/113) among males. Body mass index, fat index, Android fat, Gynoid fat, limb fat, trunk fat, and total body bone mineral content were all lower in the muscle mass loss group compared to the non-muscle mass loss group, regardless of gender (all P< 0.001). Muscle mass loss in obese adults with T2DM was affected by BMI, body fat index, and limb fat. CONCLUSION: Muscle mass loss is more prevalent in adults with T2DM and a high PBF. Body mass index, body fat index, and limb fat are the protective factors of muscle mass loss in adult patients with T2DM and obesity.

TFEB-vacuolar ATPase signaling regulates lysosomal function and microglial activation in tauopathy.

Transcription factor EB (TFEB) mediates gene expression through binding to the coordinated lysosome expression and regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase), which are essential for lysosome acidification. Single-nucleus RNA sequencing of wild-type and PS19 (Tau) transgenic mice expressing the P301S mutant tau identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and were locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.

CircMIRLET7BHG, upregulated in an m6A-dependent manner, induces the nasal epithelial barrier dysfunction in allergic rhinitis pathogenesis.

OBJECTIVE: Allergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression. METHODS: Ovalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays. RESULTS: An increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1. CONCLUSION: METTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Self-construal modulates context-based social comparison preferences in outcome evaluations.

Self-construal (SC) describes how people perceive the relationship between themselves and others and is usually divided into interdependent and independent types. Several studies have been conducted on how people with independent and interdependent SC process their own and others' outcomes. However, few studies have investigated the influence of SC on outcome evaluation in a social comparison context. To explore this, we randomly assigned participants to interdependent and independent SC priming groups and analyzed the affects and electrophysiological responses generated when they played gambling games with two pseudo-players. The results showed that self-gambling state, SC, and social comparison interacted to influence feedback-related negativity (FRN). In the self-win condition, performances that differed from others elicited more negative FRN than evenness for both the interdependent and independent groups. In the self-loss condition, this effect was only found in the independent group. These results suggest that the outcome evaluation patterns in social contexts are not fixed but vary according to self-gambling state and SC.

Biomechanical origins of proprioceptor feature selectivity and topographic maps in the Drosophila leg.

Our ability to sense and move our bodies relies on proprioceptors, sensory neurons that detect mechanical forces within the body. Different subtypes of proprioceptors detect different kinematic features, such as joint position, movement, and vibration, but the mechanisms that underlie proprioceptor feature selectivity remain poorly understood. Using single-nucleus RNA sequencing (RNA-seq), we found that proprioceptor subtypes in the Drosophila leg lack differential expression of mechanosensitive ion channels. However, anatomical reconstruction of the proprioceptors and connected tendons revealed major biomechanical differences between subtypes. We built a model of the proprioceptors and tendons that identified a biomechanical mechanism for joint angle selectivity and predicted the existence of a topographic map of joint angle, which we confirmed using calcium imaging. Our findings suggest that biomechanical specialization is a key determinant of proprioceptor feature selectivity in Drosophila. More broadly, the discovery of proprioceptive maps reveals common organizational principles between proprioception and other topographically organized sensory systems.

Brain responses to positive and negative events in individuals with internet gaming disorder during real gaming.

Objective: This study sought to investigate brain responses to positive and negative events in individuals with internet gaming disorder (IGD) during real gaming as a direct assessment of the neural features of IGD. This investigation reflects the neural deficits in individuals with IGD while playing games, providing direct and effective targets for prevention and treatment of IGD. Methods: Thirty subjects with IGD and fifty-two matched recreational game use (RGU) subjects were scanned while playing an online game. Abnormal brain activities during positive and negative events were detected using a general linear model. Functional connectivity (FC) and correlation analyses between neural features and addiction severity were conducted to provide additional support for the underlying neural features. Results: Compared to the RGU subjects, the IGD subjects exhibited decreased activation in the dorsolateral prefrontal cortex (DLPFC) during positive events and decreased activation in the middle frontal gyrus (MFG), precentral gyrus and postcentral gyrus during negative events. Decreased FC between the DLPFC and putamen during positive events and between the MFG and amygdala during negative events were observed among the IGD subjects. Neural features and addiction severity were significantly correlated. Conclusions: Individuals with IGD exhibited deficits in regulating game craving, maladaptive habitual gaming behaviors and negative emotions when experiencing positive and negative events during real game-playing compared to RGU gamers. These abnormalities in neural substrates during real gaming provide direct evidence for explaining why individuals with IGD uncontrollably and continuously engage in game playing, despite negative consequences.

Aging Fly Cell Atlas identifies exhaustive aging features at cellular resolution.

Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole aging Drosophila. We characterized 163 distinct cell types and performed an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further developed aging clock models to predict fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find distinctive cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.

Clonal hematopoiesis is associated with protection from Alzheimer's disease.

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant expansion of mutated hematopoietic stem cells. As CHIP-associated mutations are known to alter the development and function of myeloid cells, we hypothesized that CHIP may also be associated with the risk of Alzheimer's disease (AD), a disease in which brain-resident myeloid cells are thought to have a major role. To perform association tests between CHIP and AD dementia, we analyzed blood DNA sequencing data from 1,362 individuals with AD and 4,368 individuals without AD. Individuals with CHIP had a lower risk of AD dementia (meta-analysis odds ratio (OR) = 0.64, P = 3.8 × 10-5), and Mendelian randomization analyses supported a potential causal association. We observed that the same mutations found in blood were also detected in microglia-enriched fraction of the brain in seven of eight CHIP carriers. Single-nucleus chromatin accessibility profiling of brain-derived nuclei in six CHIP carriers revealed that the mutated cells comprised a large proportion of the microglial pool in the samples examined. While additional studies are required to validate the mechanistic findings, these results suggest that CHIP may have a role in attenuating the risk of AD.

Aging Atlas Reveals Cell-Type-Specific Regulation of Pro-longevity Strategies.

Organism aging occurs at the multicellular level; however, how pro-longevity mechanisms slow down aging in different cell types remains unclear. We generated single-cell transcriptomic atlases across the lifespan of Caenorhabditis elegans under different pro-longevity conditions (http://mengwanglab.org/atlas). We found cell-specific, age-related changes across somatic and germ cell types and developed transcriptomic aging clocks for different tissues. These clocks enabled us to determine tissue-specific aging-slowing effects of different pro-longevity mechanisms, and identify major cell types sensitive to these regulations. Additionally, we provided a systemic view of alternative polyadenylation events in different cell types, as well as their cell-type-specific changes during aging and under different pro-longevity conditions. Together, this study provides molecular insights into how aging occurs in different cell types and how they respond to pro-longevity strategies.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells, 1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Social value orientation modulates behavioral and neural responses to social influence.

Substantial studies have investigated the social influence effect; however, how individuals with different social value orientations (SVOs), prosocials and proselfs, respond to different social influences remains unknown. This study examines the impact of positive and negative social information on the responses of people with different SVOs. A face-attractiveness assessment task was employed to investigate the relationships between influence probability, memory, and event-related potentials of social influence. A significant interactional effect suggested that prosocials and proselfs reacted differently to positive (group rating was more attractive) and negative (group rating was less attractive) social influences. Specifically, proselfs demonstrated significantly higher influence probability, marginally better recall performance, smaller N400, and larger late positive potential on receiving negative influence information than on receiving positive influence information, while prosocials showed no significant differences. Overall, correlations between N400/LPP, influence probability, and recall performance were significant. The above results indicate the modulating role of SVO when responding to social influence. These findings have important implications for understanding how people conform and how prosocial behavior occurs.

TFEB-vacuolar ATPase signaling regulates lysosomal function and microglial activation in tauopathy.

Transcription factor EB (TFEB) mediates gene expression through binding to the Coordinated Lysosome Expression And Regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase) essential for lysosome acidification. Single nucleus RNA-sequencing (snRNA-seq) of wild-type and PS19 (Tau) transgenic mice identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. We show that the CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and was locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homoeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.

Macrophage inflammatory and regenerative response periodicity is programmed by cell cycle and chromatin state.

Cell cycle (CC) facilitates cell division via robust, cyclical gene expression. Protective immunity requires the expansion of pathogen-responsive cell types, but whether CC confers unique gene expression programs that direct the subsequent immunological response remains unclear. Here, we demonstrate that single macrophages (MFs) adopt different plasticity states in CC, which leads to heterogeneous cytokine-induced polarization, priming, and repolarization programs. Specifically, MF plasticity to interferon gamma (IFNG) is substantially reduced during S-G2/M, whereas interleukin 4 (IL-4) induces S-G2/M-biased gene expression, mediated by CC-biased enhancers. Additionally, IL-4 polarization shifts the CC-phase distribution of MFs toward the G2/M phase, providing a subpopulation-specific mechanism for IL-4-induced, dampened IFNG responsiveness. Finally, we demonstrate CC-dependent MF responses in murine and human disease settings in vivo, including Th2-driven airway inflammation and pulmonary fibrosis, where MFs express an S-G2/M-biased tissue remodeling gene program. Therefore, MF inflammatory and regenerative responses are gated by CC in a cyclical, phase-dependent manner.

Combined Detection of Islet Autoantibodies for Clinical Diagnosis of Type 1 Diabetes in the Low-Prevalence Population.

CONTEXT: Single positive islet autoantibodies (IAbs), sometimes detected in healthy individuals and patients with low-risk of developing type 1 diabetes (T1D), are considered to be irrelevant to the development of diabetes, making it difficult to diagnose and classify adult-onset diabetes. OBJECTIVE: To determine the significance and clinical value of IAbs in T1D diagnosis in the low-prevalence population, and to explore whether an electrochemiluminescence IAb detection assay can improve the clinical utility of IAbs in the immunodiagnosis of T1D in the low-prevalence population. METHODS: A total of 633 newly diagnosed patients with adult-onset diabetes (≥18 years old) were divided into 2 groups according to their clinical phenotypes: 575 patients with age at diagnosis ≥35 years and body mass index (BMI) ≥ 24 kg/m2 were considered a low-prevalence population (population with a low prevalence of T1D) and the other 58 patients were considered a high-prevalence population. All the samples from 633 participants were tested with IAbs using standard radiobinding assays (RBAs) and electrochemiluminescence (ECL) assays in parallel. RESULTS: Compared with the high-prevalence population, fewer positive IAbs (94/575, 16.3% vs 28/58, 48.3%) were detected in the low-prevalence population, and more of whom (69/94, 73.4% vs 9/28, 32.2%) were positive for a single IAb, with glutamate decarboxylase antibodies being the most prevalent single IAb. Single-IAb detection in the low-prevalence population did not always suggest the T1D phenotype. Combined detection of IAbs by RBA and ECL assay had a significant clinical utility to distinguish autoimmune diabetes in the low-prevalence population with low BMI, poor ß-cell function at the diagnosis, and an accelerated decline in ß-cell function during the follow-up. CONCLUSION: Combined autoantibody detection by RBA and ECL assays improved differentiating autoimmune from nonautoimmune diabetes in the low-prevalence population.

Exploration of Predictive Biomarkers for Postoperative Recurrence in Chronic Rhinosinusitis with Nasal Polyps Based on Serum Multiple-Cytokine Profiling.

Background: Functional nasal endoscopic surgery (FESS) is an effective treatment approach for chronic rhinosinusitis with nasal polyps (CRSwNP) patients, but some patients still suffer from postoperative recurrence. This study is aimed at investigating the expression of multiple cytokines in CRSwNP and revealing their relationships with postoperative recurrence. Methods: A total of 72 patients with CRSwNP, including 36 primary and 36 recurrent patients, were enrolled. Serum samples were obtained, 30 cytokine levels were measured by multiplex analysis, and the association between cytokine levels and recurrence was assessed. The most potential cytokines were further validated in another independent cohort with 60 primary and 60 recurrent CRSwNP patients. Results: The results of multiple cytokine profiling exhibited that the levels of eotaxin, G-CSF, IFN-α, IL-13, IL-17A, IL-5, MCP-1, and RANTES were vastly changed in the recurrent group in comparison with the primary group. Receiver-operating characteristic (ROC) curves highlighted that serum levels of eotaxin, IL-17A, and RANTES were strongly predictive of postoperative recurrence (area under the curve (AUC) > 0.7, P < 0.05). Further validation results showed that elevated serum eotaxin, IL-17A, and RANTES levels were enhanced in the recurrent group. The ROC curve showed that serum eotaxin (AUC = 0.729, P < 0.001) and RANTES (AUC = 0.776, P < 0.001) exhibited stronger ability than serum IL-17A (AUC = 0.617, P = 0.027) in predicting CRSwNP recurrence. Conclusion: Our data suggested that serum multiple cytokine profiling was associated with postoperative recurrence of CRSwNP, and eotaxin and RANTES might serve as potential biomarkers for predicting postoperative recurrence. These results might contribute to the understanding of the underlying mechanisms of recurrence and provide novel clues for precision therapy in CRSwNP.

Divergent clonal differentiation trajectories of T cell exhaustion.

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Texterm) or a killer cell lectin-like receptor-expressing cytotoxic (TexKLR) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Texterm-biased, TexKLR-biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Texterm, whereas low avidity correlates with effector-like TexKLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence.