Sensitive bispecific chimeric T cell receptors for cancer therapy.

The expression of a synthetic chimeric antigen receptor (CAR) to redirect antigen specificity of T cells is transforming the treatment of hematological malignancies and autoimmune diseases [1-7]. In cancer, durable efficacy is frequently limited by the escape of tumors that express low levels or lack the target antigen [8-12]. These clinical results emphasize the need for immune receptors that combine high sensitivity and multispecificity to improve outcomes. Current mono- and bispecific CARs do not faithfully recapitulate T cell receptor (TCR) function and require high antigen levels on tumor cells for recognition [13-17]. Here, we describe a novel synthetic chimeric TCR (ChTCR) that exhibits superior antigen sensitivity and is readily adapted for bispecific targeting. Bispecific ChTCRs mimic TCR structure, form classical immune synapses, and exhibit TCR-like proximal signaling. T cells expressing Bi-ChTCRs more effectively eliminated tumors with heterogeneous antigen expression in vivo compared to T cells expressing optimized bispecific CARs. The Bi-ChTCR architecture is resilient and can be designed to target multiple B cell lineage and multiple myeloma antigens. Our findings identify a broadly applicable approach for engineering T cells to target hematologic malignancies with heterogeneous antigen expression, thereby overcoming the most frequent mechanism of relapse after current CAR T therapies.

The detoxification ability of sex-role reversed seahorses determines the sexual dimorphism in immune responses to benzo[a]pyrene exposure.

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca2+, autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus.

Too anthropomorphized to keep distance: The role of social psychological distance on meat inclinations.

Keeping a distance from food animals helps alleviate moral conflicts associated with meat consumption. Prior research on the 'meat paradox' has shown that physical distance from animals reduces negative emotional responses when consuming meat. However, even with physical distance, the presence of animals in meat advertisements and packaging can establish psychological contact. The impact of psychological distance on meat consumption and purchase inclinations has not been well explored. Through four experiments, we discovered that animal anthropomorphism psychologically brings consumers closer to food animals, resulting in reduced intentions to consume and purchase meat. Anthropomorphized animal images notably reduced social psychological distance for consumers with moderate to high (vs. lower) levels of anthropomorphic tendencies. Furthermore, the effect of anthropomorphism was influenced by moral self-efficacy. Specifically, when social psychological distance was reduced, consumers with higher (vs. lower) moral self-efficacy exhibited a significant decrease in their willingness to consume and purchase meat. These findings expand our understanding of the role of anthropomorphism in meat marketing, its limitations, and offer insights for sales strategies. Additionally, the research could inform public health policies on meat consumption, addressing environmental and ethical concerns tied to meat production amid growing worries about animal welfare.

Effects of Simulated Microgravity on Acquired Antibiotic Resistance in Klebsiella pneumoniae Exposed to Trace Antibiotic.

Objective: The purpose of this study is to gain a better understanding of the impact of microgravity on antibiotic resistance. Methods: K. pneumoniae original (KPO) strain was cultured under either simulated microgravity (SMG) conditions with background antibiotic exposure (SMGA) for the experimental strain or a normal gravity condition with background antibiotic exposure (NGA) for the control strain. The K. pneumoniae original (KPO) strain was also cultured under normal gravity (NG) as an additional control. Antibiotic susceptibility was evaluated prior to their incubation under SMGA, NGA, or NG conditions. After 20 cycles of incubation, antibiotic susceptibility, genomic, transcriptomic, and proteomic tests were conducted on them. Results: SMGA and NGA strains both showed resistance to ciprofloxacin and intermediate resistance to levofloxacin. Genes associated with antibiotic resistance of Klebsiella pneumoniae, including acrB, oqxB, oqxA, ompC, ompF, and tolC were found to be differently expressed between SMGA and NGA strains or between SMGA and NG strains. It was found that the biggest family of genes in the differently expressed gene (DEG) cluster between SMGA and NGA and between SMGA and NG was the same, paaBCDFGHI, but with opposite change direction, i.e., downregulation between SMGA and NGA strains, while upregulation between SMGA and NG strains. Besides, the top-ranking functional descriptions in terms of the number of DEGs whether between SMGA and NGA or between SMGA and NG were "amino acid transport and metabolism", "carbohydrate transport and metabolism", "transcription", and "inorganic ion transport and metabolism". Two pathways of "citrate cycle (TCA cycle)" and "oxidative phosphorylation" were significantly enriched by DEGs both between SMGA and NGA and between SMGA and NG. Conclusion: Our study confirmed that low levels of antibiotics present in SMG can select for resistant K. pneumoniae strains. However, SMG did not alter the antibiotic resistance in K. pneumoniae induced by exposure to trace antibiotic.

Optimized Nomogram for Nasopharyngeal Carcinoma Prognosis Prediction in Younger Patients (Aged 18-59): Development and Validation.

PURPOSE: To develop a nomogram model for the predicted overall survival (OS) in patients aged 18 to 59 years with nasopharyngeal carcinoma (NPC) and assess the value of the clinical application. METHODS: In total, 1334 registers of NPC patients from 2010 to 2015 were retrieved from the Surveillance, Epidemiology, and End Results database. Univariate and multivariate Cox analysis were used to screen out independent risk factors affecting patients. Cox analysis predicted OS for patients with NPC at 3, 5, and 8 years. Nomogram performance was validated using the concordance index (C-index), receiver operating characteristic, calibration curve, and decision curve analysis (DCA). RESULTS: Age, sex, race, marital, histological type, tumor size, AJCC stage, and radiotherapy were independent risk factors. The C-index of the nomogram was 0.69 [95% confidence interval (CI): 0.68-0.71] for the training set, and the C-index of the AJCC stage was 0.63 (95% CI: 0.62-0.65), both statistically significant (P < .01). The area under the curve for the nomogram at these intervals (0.755, 0.729, and 0.729, respectively) was higher than that of the AJCC stage (0.667, 0.646, and 0.646, respectively), indicating better predictive accuracy. The calibration curves revealed a high degree of agreement between the observation and the prediction. Compared to the American Joint Committee on Cancer (AJCC) stage, DCA showed better clinical utility. CONCLUSION: The nomogram as novel predictor for nasopharyngeal carcinoma patients' survival.

The differences in plant invasion in two types of shorelines under flow regulation of the Three Gorges Reservoir.

Riparian zones, crucial for linking fluvial and terrestrial habitats, are among the most diverse ecosystems. However, they are intensively invaded by alien plants, particularly in dam-regulated rivers. Therefore, understanding the mechanisms underlying plant invasion in dam-regulated river systems has become increasingly important, given that over two-thirds of global rivers are artificially regulated. Regulated rivers may flood upland areas or pristine riparian zones, resulting in shorelines developed from pre-upland and pre-riparian areas. However, differences in invasion intensities, adaptive strategies of invasive plants, and native species' resistance (namely the diversity-invasibility relationship) across these shorelines are unclear. To address these uncertainties, we performed field investigations in the Three Gorges Reservoir (TGR) on the upper Yangtze River, where both pre-upland and pre-riparian shorelines are present. Our findings indicate that pre-upland shorelines are more intensively invaded, showing higher relative richness and cover of invasive species. Invasive plants in this area displayed more conservative resource strategies and greater drought tolerance, exhibiting lower community-weighted mean (CWM) specific leaf area, higher CWM leaf dry mass content, and larger CWM seed mass. Pre-upland shorelines' invasibility decreased as the richness and cover of native species increased, a trend not observed in pre-riparian shorelines. The observed variations in plant invasion between the two shoreline types are primarily driven by differences in resident plant presence, soil moisture levels, and hydrological disturbances. This study provides valuable insights for policymakers and practitioners involved in managing invasive plants in regulated river ecosystems.

IDR-induced CAR condensation improves the cytotoxicity of CAR-Ts against low-antigen cancers.

Chimeric antigen receptor (CAR)-T cell-based therapies demonstrate remarkable efficacy for the treatment of otherwise intractable cancers, particularly B-cell malignancies. However, existing FDA-approved CAR-Ts are limited by low antigen sensitivity, rendering their insufficient targeting to low antigen-expressing cancers. To improve the antigen sensitivity of CAR-Ts, we engineered CARs targeting CD19, CD22, and HER2 by including intrinsically disordered regions (IDRs) that promote signaling condensation. The "IDR CARs" triggered enhanced membrane-proximal signaling in the CAR-T synapse, which led to an increased release of cytotoxic factors, a higher killing activity towards low antigen-expressing cancer cells in vitro, and an improved anti-tumor efficacy in vivo. No elevated tonic signaling was observed in IDR CAR-Ts. Together, we demonstrated IDRs as a new tool set to enhance CAR-T cytotoxicity and to broaden CAR-T's application to low antigen-expressing cancers.

Biomolecular Condensation of SH2 Domain-Containing Proteins on Membranes.

The plasma membrane serves as an effective platform for signal transduction of membrane receptor pathways. Activation of the T-cell receptor (TCR) triggers the formation of membrane-associated condensates that are formed through liquid-liquid phase separation. These condensates are assembled by multivalent interactions between the tyrosine-phosphorylated receptor/adaptor and the SH2 domain-containing protein at membrane-proximal milieu. Here, we describe a biochemical reconstitution system that has been implemented to decipher the mechanisms of phospholipase PLCγ1-mediated LAT condensate formation. To characterize the interaction between specific phosphotyrosine-SH2 pair, we developed a total internal reflection fluorescence (TIRF) microscopy-based system to quantify the binding preference of each SH2 domain to specific tyrosine in the context of membranes. An assay to determine the condensate-mediated protection of phosphotyrosines from being dephosphorylated by phosphatase is also elaborated. These assays could be applied to study other transmembrane receptor pathway as well as condensates formed on endomembrane systems including the endoplasmic reticulum, mitochondrion, and Golgi apparatus.

The physical landscape of CAR-T synapse.

Chimeric antigen receptor (CAR)-T cells form dynamic immunological synapses with their cancer cell targets. After a CAR-antigen engagement, the CAR-T synapse forms, matures, and finally disassembles, accompanied by substantial remodeling of cell surface proteins, lipids, and glycans. In this review, we provide perspectives for understanding protein distribution, membrane topology, and force transmission across the CAR-T synapse. We highlight the features of CAR-T synapses that differ from T cell receptor synapses, including the disorganized protein pattern, adjustable synapse width, diverse mechano-responding properties, and resulting signaling consequences. Through a range of examples, we illustrate how revealing the biophysical nature of the CAR-T synapse could guide the design of CAR-Ts with improved anti-tumor function.

In-situ encapsulation and construction of Lac@HOFs/hydrogel composite for enhancing laccase stability and azo dyes decolorization efficiency.

Enzymes with high catalytic activity and stability have been used for the sustainable development of green chemical applications, such as water remediation. Immobilized laccase can be used to construct a synergistic system for adsorption and degradation, which has great potential for water remediation. Herein, a hydrogen-bonded organic framework was installed onto laccase in-situ to form a net-carboxylate-arranged defective cage, which enhanced its catalytic stability. Thereafter, the CMC/PVA/Lac@HOF-101 hydrogel was fabricated by freeze-thaw cycles using sodium carboxymethylcellulose and polyvinyl alcohol as carriers and copper (II) as a cross-linker. Notably, the MOFs/hydrogel as a protective carrier of laccase maintain long-term recyclability and catalytic stability. After the fifth catalytic cycle, approximately 66.7 % activity of the CP-Lac@HOF-101 was retained. When both free laccase and CP-Lac@HOF-101 were used for decolorization of Acid Orange 7 (AO), the removal rates were 10.9 % and 82.5 % after 5 h, respectively. Furthermore, even in the presence of metal cations, almost 60.0 % of the AO removal efficiency was achieved. The relationship between the structure of the azo dyes and decolorization efficiency of the synergistic system was further investigated. This study offers a method for constructing enzyme@HOF-based composite hydrogels and provides a promising water remediation strategy.

Study on the Characteristics of Early Cytokine Storm Response to Cardiac Surgery.

Cardiac surgery can provoke an acute cytokine storm that may contribute to the development of postoperative multiple organ dysfunction syndrome. We prospectively observed patients undergoing cardiac surgery and divided them into two groups: the severe group and the mild group. Healthy individuals were enrolled acting as the control group for comparison. Plasma samples and clinical data were recorded at the initiation of cardiac-pulmonary bypass (CPB) and 3, 6, 12, 24, and 48 h after initiation of CPB. Cytokine levels were detected using the Luminex® technique. Thirty-nine adults were enrolled in this study (14 in the severe group, 15 in the mild group, and 10 in the control group). Cytokine concentrations were significantly higher in the severe group. Principal component analysis was used to establish a cytokine storm intensity curve, which represented the overall trend of 10 cytokines. The peak concentrations of interleukin (IL)-6, IL-10, and IL-16 were 425.1, 198.5, and 623.0 pg/mL, which were more than 1,200, 1,800, and 240 times the normal level, respectively. The maximum cytokine storm intensity predated the maximum Vasoactive-Inotropic Score (VIS) and Sequential Organ Failure Assessment (SOFA) score in the severe group. Cytokine storm response to cardiac surgery occurred early and was associated with disease severity. Interventions to cytokine storm should be initiated early as guided by cytokine storm biomarkers such as IL-6, IL-10, and IL-16 in severe patients undergoing cardiac surgery. Clinical Trial Registration: ChiCTR1900021351.

Corrigendum: Edwardsiella piscicida infection reshapes the intestinal microbiome and metabolome of big-belly seahorses: mechanistic insights of synergistic actions of virulence factors.

[This corrects the article DOI: 10.3389/fimmu.2023.1135588.].

Effects of short-term exposure to simulated microgravity on the physiology of Bacillus subtilis and multiomic analysis.

In our study, Bacillus subtilis was disposed to a simulated microgravity (SMG) environment in high-aspect ratio rotating-wall vessel bioreactors for 14 days, while the control group was disposed to the same bioreactors in a normal gravity (NG) environment for 14 days. The B. subtilis strain exposed to the SMG (labeled BSS) showed an enhanced growth ability, increased biofilm formation ability, increased sensitivity to ampicillin sulbactam and cefotaxime, and some metabolic alterations compared with the B. subtilis strain under NG conditions (labeled BSN) and the original strain of B. subtilis (labeled BSO). The differentially expressed proteins (DEPs) associated with an increased growth rate, such as DNA strand exchange activity, oxidoreductase activity, proton-transporting ATP synthase complex, and biosynthetic process, were significantly upregulated in BSS. The enhanced biofilm formation ability may be related with the DEPs of spore germination and protein processing in BSS, and differentially expressed genes involved in protein localization and peptide secretion were also significantly enriched. The results revealed that SMG may increase the level of related functional proteins by upregulating or downregulating affiliated genes to change physiological characteristics and modulate growth ability, biofilm formation ability (epsB, epsC, epsN), antibiotic sensitivity (penP) and metabolism. Our experiment may gives new ideas for the study of space microbiology.

Edwardsiella piscicida infection reshapes the intestinal microbiome and metabolome of big-belly seahorses: mechanistic insights of synergistic actions of virulence factors.

Uncovering the mechanism underlying the pathogenesis of Edwardsiella piscicida-induced enteritis is essential for global aquaculture. In the present study, we identified E. piscicida as a lethal pathogen of the big-belly seahorse (Hippocampus abdominalis) and revealed its pathogenic pattern and characteristics by updating our established bacterial enteritis model and evaluation system. Conjoint analysis of metagenomic and metabolomic data showed that 15 core virulence factors could mutually coordinate the remodeling of intestinal microorganisms and host metabolism and induce enteritis in the big-belly seahorse. Specifically, the Flagella, Type IV pili, and Lap could significantly increase the activities of the representative functional pathways of both flagella assembly and bacterial chemotaxis in the intestinal microbiota (P < 0.01) to promote pathogen motility, adherence, and invasion. Legiobactin, IraAB, and Hpt could increase ABC transporter activity (P < 0.01) to compete for host nutrition and promote self-replication. Capsule1, HP-NAP, and FarAB could help the pathogen to avoid phagocytosis. Upon entering epithelial cells and phagocytes, Bsa T3SS and Dot/Icm could significantly increase bacterial secretion system activity (P < 0.01) to promote the intracellular survival and replication of the pathogen and the subsequent invasion of the neighboring tissues. Finally, LPS3 could significantly increase lipopolysaccharide biosynthesis (P < 0.01) to release toxins and kill the host. Throughout the pathogenic process, BopD, PhoP, and BfmRS significantly activated the two-component system (P < 0.01) to coordinate with other VFs to promote deep invasion. In addition, the levels of seven key metabolic biomarkers, Taurine, L-Proline, Uridine, L-Glutamate, Glutathione, Xanthosine, and L-Malic acid, significantly decreased (P < 0.01), and they can be used for characterizing E. piscicida infection. Overall, the present study systematically revealed how a combination of virulence factors mediate E. piscicida-induced enteritis in fish for the first time, providing a theoretical reference for preventing and controlling this disease in the aquaculture of seahorses and other fishes.

Anti-tumor Efficacy of CD19 CAR-T in a Raji B Cell Xenografted Mouse Model.

Chimeric antigen receptor (CAR)-T therapy launched a new era for cancer treatments, displaying outstanding effectiveness in relapsed or refractory B-cell malignancies. Demonstrating the tumor-killing ability of CAR-Ts in mouse xenograft models serves as a golden criterium in preclinical research. Here, we describe a detailed method for evaluating CAR-T's function in immune-deficient mice bearing Raji B cell-induced tumors. It includes generating CD19 CAR-T cells from healthy donors, injecting tumor cells and CAR-T cells into mice, and monitoring tumor growth and CAR-T state. This protocol provides a practical guide to evaluate CAR-T's function in vivo within eight weeks. Graphical abstract.

Imaging CAR-T Synapse as a Quality Control for CAR Engineering.

The chimeric antigen receptor (CAR) evolves as a powerful tool to reprogram T cells for targeted killing. CAR-T therapy succeeded in treating certain types of blood cancers, and its application is now expanding towards solid tumors, autoimmune diseases, viral infection, and fibrosis. These require the design of a large number of new CARs that target a variety of antigens. Here we described two methods as a quality control for validating newly developed CARs: (1) the cell-cell conjugation assay as a reflection of efficient binding of CAR to antigen in the cellular context and (2) CD45 exclusion in the synapse as an indication of CAR signaling potential. These assays examine prerequisites for a functional CAR-T and reveal causes for ineffective CAR-T activation.

Functional exploration of SNP mutations in HIF2αb gene correlated with hypoxia tolerance in blunt snout bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A203A752) and its homozygous diplotype II (A203A203A752A752) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOEcrit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48 h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48 h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.

Effects of hypoxia and reoxygenation on oxidative stress, histological structure, and apoptosis in a new hypoxia-tolerant variety of blunt snout bream (Megalobrama amblycephala).

A new hypoxia-tolerant variety of blunt snout bream was obtained by successive breeding of the wild population, which markedly improved hypoxia tolerance. In this study, the hypoxia-tolerant variety was exposed to hypoxia (2.0 mg O2·L-1) for 4, 7 days. The contents of blood biochemical indicators including the number of red blood cells (RBC), total cholesterol (T-CHO), total protein (TP), triglyceride (TG), glucose (GLU), and lactic acid (LD) increased significantly (P < 0.05) under hypoxia. The glycogen content in the liver and muscle decreased significantly (P < 0.05) and the LD content in the brain, muscle and liver increased significantly (P < 0.05) under hypoxia. The levels of oxidative stress-related indicators i.e., superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) also changed significantly (P < 0.05) in the heart, liver, and intestine of the new variety under hypoxia. Additionally, hypoxia has caused injuries to the heart, liver, and intestine, but it shows amazing repair ability during reoxygenation. The apoptotic cells and apoptosis rate in the heart, liver, and intestine increased under hypoxia. Under hypoxia, the expression of the B-cell lymphomas 2 (Bcl-2) gene in the heart, liver, and intestine was significantly (P < 0.05) down-regulated, while the expression of the BCL2-associated agonist of cell death (Bad) gene was significantly (P < 0.05) up-regulated. These results are of great significance for enriching the basic data of blunt snout bream new variety in response to hypoxia and promoting the healthy development of its culture industry.

Efficacy of first-line systemic treatment regimens for recurrent/metastatic head and neck squamous cell carcinoma: a network meta-analysis.

PURPOSE: To evaluate the efficacy on overall survival (OS) and progression-free survival (PFS) of the first-line systemic therapy regimens on 6-, 12-, 18-, 24-, and 30 months in recurrent/metastatic head and neck squamous cell carcinoma (R/M-HNSCC) and figure out the best regimen. METHODS: PubMed, Embase, The Cochrane Library, Scopus, and Google Scholars were systematically searched for studies in regard to the first-line systemic regimens for R/M-HNSCC from inception to March 2022. Odds ratios (ORs) were generated for dichotomous variants by network meta-analysis. The primary endpoint was OS, and the second endpoint was PFS. The software implemented was STATA 17.0 MP. RESULTS: Eventually, 18 studies with 5298 patients and 12 first-line systematic regimens were enrolled. immunotherapy + chemotherapy (OR = 2.30, 95% CI 1.60-3.31) and single immunotherapy (OR = 1.91, 95% CI 1.33-2.76) were significantly superior to the EXTREME on OS at 30th month. Meantime, immunotherapy + chemotherapy (SUCRA = 87.7%) has the highest ranking. TPEx (OR = 1.61, 95% CI 1.05-2.48) showed significantly better efficacy compared with EXTREME on PFS at 12th month. Simultaneously, TPEx (SUCRA = 87.1%) had the highest ranking and was the long-lasting first-echelon regimen both in OS and PFS from a longitudinal perspective. It should be noted that EXTREME included platinum-based chemotherapy + fluorouracil + cetuximab, TPEx included docetaxel + cisplatin + cetuximab. CONCLUSION: Considering the efficacy, safety, compliance, and economic profiles collectively, one of the standard first-line regimens, literally TPEx should be recommended as the best choice for R/M-HNSCC. Furthermore, more head-to-head trials are needed to confirm those findings.

Size-dependent activation of CAR-T cells.

As the targets of chimeric antigen receptor (CAR)-T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a "size exclusion" model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.