顶刊文献解读｜深挖细胞稳态失衡介导疾病发生的高分机制突破

Figure 1. Lactic acid upregulates the expression level of TXNIP. Healthy human T cells (n=3) were expanded in vitro and treated with 25?mM lactic acid, with samples collected at 0.5, 1, and 3?hours, as well as 1-hour transition to neutral condition following a 3-hour treatment with lactic acid. The blank control group was cultured in lactic acid-free TGM for 3?hours before sampling.

(A) Heatmap analysis of eight upregulated genes derived from RNA-seq data.

(B) Extraction of mRNA for RT-qPCR to assess the mRNA expression level of the ZNF627 and TXNIP genes, with ACTB used as the internal control.

(C) T cells were activated by anti-CD3/28 dynabeads in the presence or absence of lactic acid for 6?hours, followed by Western blot analysis to detect TXNIP protein level, with β-actin serving as the internal control.

(D) The promoter prediction websites BDGP and FPROM were utilized to analyze Pfull, indicating potential transcription start sites along with their scores.

(E) Based on the prediction results, truncated variants P1, P2, and P3 corresponding to the top three scores were constructed and cloned into a lentiviral expression vector upstream of the reporter gene mCherry to establish the reporter system.

(F) HEK-293T cells were seeded at a density of 2×105?in 24-well Plates were allowed to adhere overnight. 1?μg of the reporter plasmids was then transiently transfected, and mCherry expression was visualized with fluorescence microscopy 48 hours later.

(G, H) Healthy human PBMCs were stimulated with anti-CD3/28 dynabeads (n=3). After 72 hours, the activated T cells were transduced with lentiviruses containing the reporter plasmids and subsequently expanded in vitro. On day 7 post-transduction, T cells were incubated in TGM with 15, 20, and 25?mM lactic acid, while the blank control group was in lactic acid-free medium. (G) MFI of mCherry+ cells was measured after treatment periods of 6, 12, 24, 48, and 72 hours. (H) The mCherry expression in each group was normalized against the blank control (0?mM), demonstrating fold changes in response to different concentrations of lactic acid.

The results are displayed as the mean±SEM, analyzed using a one-way ANOVA test, and comparisons are shown between the lactic acid-treated and control groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells; TGM, T cell growth medium.

Figure 2. The LARP modulates CAR expression in response to lactate regulation.

(A) Scheme of the sequence structure of mCherry reporter plasmids driven by miniE, E68, or miniE-E68.

(B–D) Healthy human PBMCs (n=3) were stimulated with anti-CD3/28 dynabeads. After 72 hours, activated T cells were transduced with lentiviruses containing the reporter plasmids and subsequently expanded in vitro. On day 7 post-transduction, T cells were cultured in either neutral or acidic conditions (with or without 25?mM lactic acid in the TGM) and collected at the indicated time points for flow cytometry analysis of mCherry expression. (B) Representative flow cytometry data showing mCherry expression levels at 48 hours. (C) MFI of mCherry+ cells at different time points. (D) The mCherry expression in each group was normalized against the basal level, illustrating fold changes following treatment with various concentrations of lactic acid.

(E) Schematic representation of the lentiviral construct of Conventional CAR driven by the constitutive SFFV promoter and LAR CAR. The HER2 CAR contained a FLAG tag for the detection of CAR expression.

(F-I) Lentiviruses were packaged using Conventional CAR and LAR CAR plasmids to transduce T cells (n=3) activated for 72 hours, followed by cultivation and expansion for around 10 days to obtain CAR-T cells. The LAR CAR-T and Conventional CAR-T cells were cultured in TGM with 15, 20, and 25?mM lactic acid. After 72 hours of treatment, some cells were transferred to a lactic acid-free TGM for another 72-hour incubation. The blank control group was kept in lactic acid-free TGM (0?mM). Cells were collected every 24 hours for flow cytometry analysis. (F) Proportion of EGFP+ cells representing the positive transduction rate of the lentivirus. (G, H) The proportion and MFI of CAR+ among EGFP+ cells. (I) The CAR expression in each group from (H) was normalized against the blank control, showing fold changes following lactic acid treatment.

The results are displayed as the mean±SEM, analyzed using a one-way ANOVA test, and comparisons are shown between the lactic acid-treated and control groups. *p<0.05, **p<0.01, ****p<0.0001. ANOVA, analysis of variance; LARP, lactic acid-responsive promoter; MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells; SFFV, spleen focus forming virus; TGM, T cell growth medium.

Figure 3. In vitro phenotypic characteristics of LAR CAR-T cells.

(A) Proliferation dye labeling was performed on conventional CAR-T and LAR CAR-T cells, which were then cultured in neutral or acidic conditions (without or with different concentrations of lactic acid in the TGM) for 2, 4, and 7?days, after which cells were collected for flow cytometry analysis of the decay in the proliferation dye intensity.

(B, C) After an 11-day culture period in neutral condition (without lactic acid), both Conventional CAR-T and LAR CAR-T cells (n=3) were stained with anti-CD3, anti-CD8, anti-DYKDDDDK Tag, anti-CD95, anti-CD45, and anti-CCR7 antibodies. (B) Proportion of CD4+ and CD8+ T cells in Conventional CAR-T and LAR CAR-T cells. (C) Gating strategy and statistical chart depicting the proportion of each effector memory phenotype of Conventional CAR-T and LAR CAR-T cells.

(D-F) To evaluate exhaustion markers, CAR-T cells cultured under the same neutral condition (without lactic acid) were stained with anti-DYKDDDDK Tag, anti-PD-1, anti-LAG-3, and anti-TIM-3 antibodies. The proportions of PD-1+ (D), LAG-3+ (E), and TIM-3+ (F) cells were analyzed in conventional CAR-T and LAR CAR-T, respectively (n=4).

(G, H) For analysis of ROS and MMP, conventional CAR-T and LAR CAR-T cells (n=3) were labeled with fluorescent probes dihydroethidium (DHE) or tetramethylrhodamine (TMRE) following incubation in neutral or acidic condition (the acidic TGM contained specified concentrations of lactic acid). The O2? production (G) and MMP levels (H) were measured by flow cytometry at 24 and 48 hours for both CAR-T cells.

The results are displayed as the mean±SEM, analyzed using Student’s t-test. *p<0.05. ns, not significant. MMP, mitochondrial membrane potential; TGM, T cell growth medium.

Figure 4. The tumor-killing activity of LAR CAR-T cells exhibited lactic acid dependency (n=3).

(A) Schematic diagram of the in vitro cytotoxicity assay workflow. Target cells (NCI-H292 or SKOV3) were plated at a density of 1×104?cells in 96-well flat-bottom plates and allowed to adhere overnight. After a 24-hour preincubation in neutral or acidic (25?mM lactic acid) condition, Conventional CAR-T and LAR CAR-T cells were then co-cultured with the target cells at specified effector-to-target (E:T) ratios (2:1, 4:1) for 2, 4, and 6?hours.

(B, C) Supernatants were collected, and cytotoxicity of CAR-T cells against NCI-H292(B) or SKOV3 (C) tumor cells was assessed using CCK-8.

The results are displayed as the mean±SEM, analyzed using Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

Figure 5. LAR CAR-T cells did not cause acute hepatotoxicity and inflammatory infiltration.

(A) Experimental workflow to assess the OTOT of LAR CAR-T therapy in B-NDG mice (n=3–4): A replication-defective recombinant adenovirus carrying HER2 antigen (Ad5-HER2) was injected intraperitoneally at a dose of 3×109?PFU to infect the mouse liver and induce the expression of the human HER2 antigen. Eight days postinfection, mice were randomly assigned to groups and received intravenous infusions of effector T cells, including UTD, LAR CAR-T cells, and Conventional CAR-T cells, as well as a solvent control (Mock group received PBS), with a volume of 125?μL for each infusion. Additionally, a group of mice uninfected with Ad5-HER2 received Conventional CAR-T cells. Body weights were measured every 2?days, and peripheral blood was collected to isolate serum samples. On days 3, 7, and 11, mice were sacrificed for tissue collection. Liver tissues were fixed with formaldehyde and prepared as paraffin sections.

(B) Change in body weight over the treatment period for each group of mice.

(C) Survival curves for each group of mice during the treatment.

(D, E) Changes in serum levels of AST and ALT 5?days post-infusion.

(F) A CBA kit was used to measure T cell activation-related cytokines in the serum of HER2 antigen-humanized mice, including IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A, to assess the immune response elicited by Conventional CAR-T and LAR CAR-T cells in vivo.

(G-I) Mice were sacrificed, and tissues were collected 11 days after the infusion therapy. Liver tissues were fixed with formaldehyde and prepared as paraffin sections. (G) IHC staining was performed to evaluate how CAR-T cell infusion affected the expression of HER2 in the liver tissues. (H) HE staining was conducted to assess the extent of inflammatory infiltration in the liver. (I) IF staining was used to detect CAR-T cell infiltration in the liver tissues, using UTD as a control.

The results are displayed as the mean±SEM, analyzed using a one-way ANOVA test, and comparisons are shown between the Conventional CAR and LAR CAR groups. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001. ns, not significant. ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; CBA, Cytometric Bead Array; IF, immunofluorescence; IHC, immunohistochemical; OTOT, on-target, off-tumor toxicity; UTD, Untransduced T cells.

Figure 6. LAR CAR-T cells exhibited in vivo antitumor activity without OTOT risks.

(A) Experimental design for simultaneous evaluation of the in vivo OTOT and anti-tumor efficacy of LAR CAR-T cells: 5×106 human ovarian cancer cells (SKOV3) were subcutaneously (s.c.) injected into B-NDG mice (n=4). 6?days later, 3×10? PFU of Ad5-HER2 was administered intraperitoneally (i.p.) to the tumor-bearing mice. After 8?days, the mice were randomly assigned to groups and received intravenous or i.p. injection of effector T cells, including UTD, conventional CAR-T cells, and different doses of LAR CAR-T cells.

(B) SKOV3 tumor growth curves.

(C) Change in body weight over the treatment period for each group of mice.

(D) Survival curves of different groups.

(E) Measurement of serum levels of AST and ALT at designated time points following CAR-T infusion.

(F) Comparison of T cell activation-related cytokines, including IL-2, IL-10, IFN-γ, and TNF-α, in the peripheral blood among the treatment groups.

The results are displayed as the mean±SEM, analyzed using a one-way ANOVA test, and comparisons are shown between the conventional CAR and LAR CAR (1.5×107) groups. *p<0.05, **p<0.01, ****p<0.0001. ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; OTOT, on-target, off-tumor toxicity; PFU, plaque-forming unit; UTD, Untransduced T cells.

【免责声明】原文献《J Immunother Cancer》（DOI：10.1136/jitc-2025-013672），由 AI 解读整理;文中涉及的原文献图片、数据等知识产权归原期刊及研究团队所有。若存在侵权情形，敬请及时联系我方删除，我方将积极配合处理。