Intrinsic ADRB2 inhibition improves CAR-T cell therapy efficacy against prostate cancer

Abstract

Chimeric antigen receptor (CAR)-T cell therapy has shown limited success in patients with solid tumors. Recent in vitro and in vivo data have shown that adrenoceptor beta-2 (ADRB2) is a novel checkpoint receptor that inhibits T cell-mediated anti-tumor responses. To inhibit ADRB2-mediated inhibitory signaling, we downregulated ADRB2 in CAR-T (shβ₂-CAR-T) cells via RNA interference, assessed different parameters, and compared them with conventional second-generation CAR-T cells. ADRB2 knockdown CAR-T cells exhibited enhanced cytotoxicity against prostate cancer cell lines in vitro, by increasing CD69, CD107a, GzmB, IFN-γ, T-bet, and GLUT-1. In addition, ADRB2 deficiency led to improved proliferation, increased CD8/CD4 T cell ratio, and decreased apoptosis in CAR-T cells. shβ₂-CAR-T cells expressed more Bcl-2 and led to the generation of more significant proportions of T central memory cells. Finally, the ZAP-70/NF-κB signaling axis was shown to be responsible for the improved functions of novel CAR-T cells. In tumor-bearing mice, shβ₂-CAR-T cells performed better than conventional CAR-T cells in eradicating prostate tumors. The study provides the basis for future clinical and translational CAR-T cell research to focus on adrenergic stress-mediated challenges in the tumor microenvironment of stressed tumors.

Keywords: ADRB2; CAR; CAR-T cell therapy; adrenergic stress; adrenoceptor beta-2; chimeric antigen receptor; combination therapy; immune checkpoint; immunotherapy; neuroimmunology; prostate cancer; tumor microenvironment.

Graphical abstract

Figure 1

Development and screening of ADRB2-shRNA (A–C) Unstimulated or CD3/CD28^– T cells (2 × 10⁶) were collected immediately after T cell isolation from PBMCs. Stimulated or (CD3/CD28)⁺ T cells were collected following 48 h of T cell activation. T cells were transduced with CAR lentivirus for 48 h, and CAR-T cells were collected. CAR-T cells were cocultured with PC-3 cells at an effector-to-target (E:T) ratio 1:1 for 4 h. Cells were collected from each experimental group. ADRB2 gene expression was analyzed by qPCR (A) and RT-PCR (B). (C) Flow cytometry analysis of ADRB2 in each experimental group. (D) Schematic representation of ADRB2 shRNA containing pLL3.7 lentiviral plasmids. (E–G) Primary T cells were transduced with shADRB2-A, shADRB2-B, shADRB2-C, or shADRB2-NC lentiviruses (MOI = 10) and cultured for 48 h. (E) The cells were harvested and subjected directly to the flow cytometry. eGFP protein expression was measured and depicted as the transduction efficiency of T cells. Gene expression analysis of ADRB2. (F and G) 2 × 10⁶ transduced T cells from each group were utilized for total mRNA extraction (F) qPCR and (G) reverse transcription PCR (RT-PCR) analysis. GAPDH served as a reference and expression levels were assessed using the 2^ΔΔCT method for qPCR and gel electrophoresis for RT-PCR. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 2

Construction and screening of novel CAR-transduced T cells (A) Schematic representation of the CAR, shNC-CAR, and shβ₂-CAR vectors. (B) Working of the novel CAR construct. (C) FACS analysis of infection rate. CAR expression in transduced T cells was analyzed by FACS using an APC-conjugated anti-human NKG2D antibody at various time points. (D) qPCR analysis of ADRB2 gene expression in the experimental cohorts. The M-T cohort was used as a control. (E) Western blot analysis of the ADRB2 protein. The relative band intensity is depicted in the lower panel in graphical form. (F) qPCR analysis of ADRB2 for stable downregulation. shβ₂-CAR-transduced T cells were cultured for 8 days. Cells were collected every 2 days. RNAs were extracted and subjected to qPCR analysis. GAPDH was used as a reference. (G and H) The cells in the experimental cohorts were stained with either APC-conjugated anti-human CD69 or PE/Cy7-conjugated anti-human granzyme B antibody and subsequently analyzed via FACS. (I) The cells (0.5 × 10⁶) were stained with CFSE and analyzed via FACS on day 0 and day 5. (J) Graphical representation of the CD8/CD4 ratio. Cells were stained with BV421-conjugated anti-human CD3, FITC-conjugated anti-human CD4, and APC-conjugated anti-human CD8 antibodies and analyzed via FACS. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 3

shβ₂-CAR-T cells exhibited enhanced activation and proliferation via GLUT-1 upregulation (A) Flow cytometry analysis for NKG2D ligands. Prostate cancer cell lines (PC-3 and DU-145), non-cancerous prostate cell line (PNT), and primary human T cells were subjected to flow cytometry after staining with PE-conjugated anti-human MICA/B antibody. (B) ELISA analysis of prostate cancer cell lines (PC-3 and DU-145) for N.E concentrations. (C) The cells of experimental groups (0.5 × 10⁶) were cocultured with PC-3 cells (E:T ratio of 3:1). After 8 h, the medium was removed, and cells were stained with an APC-conjugated anti-human CD69 antibody for FACS analysis. (D) Experimental groups were stained with CFSE dye, cocultured with target cells (E:T ratio of 1:1), and analyzed by FACS on day 0 and day 5. (E) The cells were collected on day 5, stained with an APC-conjugated anti-human Ki67 antibody, and subjected to FACS analysis. (F) The cells in the experimental cohorts were stained with BV421-conjugated anti-human CD3, FITC-conjugated anti-human CD4, and APC-conjugated anti-human CD8 antibodies and analyzed via FACS. The right panel shows a graphical representation of the CD8/CD4 ratio. (G) Effector T cells were cultured with target cells for 24 h in an adherent 96-well plate. After 24 h, T cells were carefully collected, and subjected to qPCR analysis for GLUT-1 expression. (H) qPCR analysis of PGC-1α gene expression, whereas GAPDH was used as a reference. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 4

shβ₂-CAR-T cells showed enhanced cytotoxicity against prostate cancer cell lines in vitro (A) Flow cytometry-based CAR-T cell cytotoxic assay. CFSE-labeled PC-3 cells were cocultured with experimental cohorts and, after 16 h, cells were stained with anti-Annexin V antibody and subjected to flow cytometry. (B and C) Cytotoxicity analysis by CCK8 cell viability assay. The experimental cohorts were coincubated with PC-3 or DU-145 cells (E:T ratio of 1:1, 3:1, 9:1) for 16 h. CCK8 reagent was added, and the absorbance was measured at 450 nm by a microplate reader. ∗ indicates CAR-T vs. shβ₂-CAR-T cell groups, whereas # indicates M-T vs. CAR-T cells. (D) Cytotoxicity analysis by luciferase assay. The experimental cohorts were cocultured with TH-deficient PC-3 cells at various E:T ratios. Luciferase reagent was added in each sample and absorbance was measured at 560 nm by luminometer. (E–H) The experimental groups were cocultured with PC-3 target cells for 24 h. The cells were collected and stained with the appropriate FACS antibodies to detect markers related to cytotoxicity. (E) Surface CD107a expression was analyzed by staining cells with a PE/Cy7-conjugated anti-human CD107a antibody. (F) Intracellular GzmB expression analysis by staining cells with PE/Cy7-conjugated anti-human granzyme B antibody after cell membrane permeabilization. (G) Intracellular IFN-γ expression analysis by staining cells with an APC-conjugated anti-human IFN-γ antibody after cell membrane permeabilization. (H) Intranuclear T-bet expression analysis by staining cells with a PE-conjugated anti-human T-bet antibody after nuclear membrane permeabilization. FACS data are shown on the left panel, while percentage and MFI are illustrated in chat bars on the right. The ∗ and # indicate the p values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001).

Figure 5

shβ₂-CAR-T cells displayed improved cell survival and T cell memory formation (A and B) M-T, CAR-T, shNC-CAR-T, and shβ₂-CAR-T cells were cocultured with PC-3 cells at an E:T ratio of 3:1 for 3 days. Following incubation, cells were washed and stained with (A) PE-conjugated anti-human PD-1 or (B) APC-conjugated anti-human TIM3 antibodies for 30 min, and their surface expression was quantified using flow cytometry. (C) Luciferase cytotoxicity assay at 72 h. Effector cells and luciferase-containing target cells (PC-3) were cocultured at an E:T ratio of 3:1 for 3 days and OD was measured at 560 nm. (D) Trypan blue cell exclusion method for analyzing proliferation. Forty thousand cells from each experimental cohort were cultured for 7 days and after every 2 days cells were calculated using trypan blue cell exclusion method. (E) Bcl-2 expression within CD3⁺, CD4⁺, and CD8⁺ T cells was measured using flow cytometry. In brief, after 5 days of coculture, cells were stained with surface BV421-conjugated anti-human CD3, FITC-conjugated anti-human CD4, and PE/Cy7-conjugated anti-human CD8 antibodies followed by membrane permeabilization and intracellular staining with PE-conjugated anti-human Bcl-2 antibody. (F) After 5 days of coculture, cells were collected and stained with Annexin V in the dark at room temperature for 15 min, and T cell survival was assessed using flow cytometry. (G) Cells were stained with BV421-conjugated anti-human CD3, FITC-conjugated anti-human CD4, PE/Cy7-conjugated anti-human CD8, PE-conjugated anti-human CD45RO, and APC-conjugated anti-human CD62L antibodies for 30 min and subjected to flow cytometry. CD4 and CD8 T cell subsets were measured in CD3⁺ gated cells, and among CD3, CD4, and CD8 T cells, dual-color fluorescence of CD62L and CD45RO was measured. FACS files on the left illustrate distinct T cell populations, while the bar chat on the right side quantifies the percentage of T_CM. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 6

shβ₂-CAR-T cells improved pZAP-70/NF-κB signaling (A and B) Effector cells (2 × 10⁵) from each experimental group were cocultured with or without PC-3 target cells at an E:T ratio of 6:1 in a 96-well plate for 8 h. (A) ZAP-70 phosphorylation analysis without target cells. (B) ZAP-70 phosphorylation analysis with target cells (PC-3). (C and D) To detect phosphorylation of p65 the cells were stained with PE-conjugated anti-human p-P65 antibody and subjected to FACS analysis. (C) Flow cytometry analysis for p-P65 in experimental cohorts without target cells. (D) Flow cytometry analysis for p-P65 in experimental cohorts in the presence of target cells. (E–J) Target and effector cells were coincubated in a 48-well plate. The NF-κB inhibitor (QNZ) was added in the shβ₂-CAR+QNZ cohort. The cells were collected and stained with (E) APC-conjugated anti-human CD69 antibody, (F) PE/Cy7-conjugated anti-human CD107a antibody, (G) PE/Cy7-conjugated anti-human GzmB antibody, (H) APC-conjugated anti-human IFN-γ antibody, and (I) PE-conjugated anti-human PD-1 antibody to detect the expression analysis of these markers via flow cytometry. (J) M-T, CAR-T, shNC-CAR-T, shβ₂-CAR-T, and shβ₂-CAR-T + QNZ cells were cocultured with 30,000 luciferase-transduced target cells at various effector-to-target ratios (1:1, 3:1, and 9:1) for 8 h in a low adherence 96-well plate. The cells were collected, and absorbance was measured at 560 nm. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 7

shβ₂-CAR-T cells suppressed tumor growth in the PC-3 xenograft model (A) Schematic representation of the mouse experiment. NSG mice were subcutaneously inoculated with 2 × 10⁶ PC-3 cells on day 21, followed by tail vein injection of 5 × 10⁶ M-T cells, 5 × 10⁶ CAR-T cells, and 5 × 10⁶ shβ₂-CAR-T cells (n = 7) on day 0. (B) Tumor growth was monitored through in vivo bioluminescence imaging on designated days. (C–F) Average radiance was measured by total bioluminescence signals among the indicated groups at various time points. (G) Tumor volume was measured using Vernier calipers every 10 days post-CAR-T injection. (H) Overall survival of mice was depicted in Kaplan-Meier curves up to day 50. (I) Tumor tissues were excised from each group (n = 5) stained with BV421-conjugated anti-human CD3 antibody and analyzed by flow cytometry. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).

Figure 8

Circulating shβ₂-CAR-T cells displayed superior phenotypes in PC-3 mice xenograft On day 41, 100 μL of blood was collected from each mouse via the retro-orbital method and treated with red blood lysis buffer for 10 min. Following washing, cells were stained with specific FACS antibodies. (A) PE-conjugated anti-human PD-1 and (B) APC-conjugated anti-human TIM3 antibodies were utilized for expression analysis of PD-1 and TIM3, respectively. (C and D) In a separate experiment, cells were stained with surface APC-conjugated anti-human CD4, PE-conjugated anti-human CD8, APC-conjugated anti-human CD62L, and FITC-conjugated anti-human CD45RO antibodies, and the dual color fluorescence was measured. (C) CD8 and CD4 T cell subsets were visualized in a two-parameter dot plot (left side), with the CD8/CD4 ratio indicated on the right side. (D) T cell differentiation was assessed in a two-parameter zebra plot, and %T_CM is shown on the right side. p-Value (∗p < 0.05, p∗∗ < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001) and ns indicates not significant (p > 0.05).