Cell lines and cell culture conditions

Cell lines were cultured according to the manufacturers’ recommendations. NALM-6 is a pre-B cell acute lymphoblastic leukemia (ALL) cell line with high expression of CD19 (German DSMZ cell collection Cat#: ACC128). NALM-6-PDL1-GFP-luciferase (luc) is a stable cell line engineered to express PDL1-GFP-luc. K562 is a chronic myelogenous leukemia cell line (ATCC; Cat#: CCL-243). K562-CD19, K562-PDL1-GFP-luc, and K562-CD19-PDL1-GFP-luc are stable cell lines engineered to express CD19 and PDL1-GFP-luc. 786o is a renal cell adenocarcinoma cell line (ATCC; Cat#: CRL-1932™) that naturally expresses PDL1. CD19 was transduced into 786o using a lentivirus system to produce 786o-CD19. The aforementioned tumor cells were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS) 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, and 1% L-glutamine. T cells were cultured in X-VIVO15 (Lonza, USA) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin sulfate, 1% L-glutamine, and 200 U/mL IL-2. The expression of CD19 and PDL1 of tumor cells is shown in sFig.1.

Generation of CAR constructs

The conventional second-generation CAR (2G) structure in this study was constructed by fusing CD19 scFv, CD8α hinge and TMD, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. The CAR with a PD1CD28 switch-receptor (PD1-S28) structure was created by fusing the second CAR with the “PD1CD28” chimeric receptor. The PE8HT CAR-T structure was formed by fusing CD19 scFv, a linker (G4S)4, a truncated extracellular PD1 (AA21-155), CD8 hinge and TMD, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. The PEPT CAR-T structure was generated by fusing CD19 scFv, a linker (G4S)4, a truncated extracellular and transmembrane PD1 (AA21-191) derived from PD1 cDNA, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. Finally, the PE8T structure was created by fusing CD19 scFv, a linker (G4S)4, a truncated extracellular PD1 (AA21-155) derived from PD1 cDNA, CD8 TMD, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. These CAR structures were subcloned into viral vectors for transfection into activated T cells.

Production of lentivirus particles

Lentiviruses were produced by transiently transfecting three plasmids into 293T cells using Lipo2000 (Invitrogen, USA). Briefly, 80% confluent 293T cells in 15-cm plates (Nalgene Nunc, USA) were transfected with approximately 30 µg of three plasmids, comprising 5 µg of the structural plasmid pHDH-Hgpm2 (HIV gag-pol), pMD-tat, pRC/CMV-rev, and Env VSV-G, and 10 µg of the vector encoding plasmid. Following a previously published protocol, the virus supernatant was concentrated using ultracentrifugation (Backman, USA) [29]. The concentrated virus was stored at − 80 °C.

Selection, activation, and lentivector transduction of CD3 + T cells

Blood samples from healthy volunteers were obtained using an approved protocol by the Fifth Medical Center Ethics Committee of Chinese PLA General Hospital (Ethical code: Ky-2018-5-37), following the Declaration of Helsinki. All participants provided written informed consent before participating in the study. Human peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Paque PLUS. T cells were isolated using positive selection and stimulated with CD3/CD28 Dynabeads. After 48 h, activated T cells were transduced with lentivirus at a MOI of 5–10 and cultured in X-VIVO15 with 100 U/mL penicillin, 100 U/mL streptomycin sulfate, 1% L-glutamine, and 200 U/mL IL-2. The transduction efficiency for CAR-positive cells was determined by flow cytometry using a biotinylated human CD19 protein (Acrobiosystems, Cat. No. CD9-HF251, USA).

Binding assay

To evaluate the function of PE-CARs and understand the underlying mechanisms, we used 2G and PD1-S28 CARs as controls [14]. To determine the affinity of different CAR-T cells for CD19 protein, we measured the fluorescence intensity of CAR-T cells at various concentrations of CD19 protein. The number of CAR-positive and mock-T cells was consistent with that of 2G CAR-T cells. Specifically, mock-T, 2G, and PEPT CAR-T cells were washed twice with PBS (1% BSA) by centrifugation. They were then treated with CD19-Fc protein (11,880-H02H) at different final concentrations (ranging from 180 µg/mL to 0.05 µg/mL). The cells were incubated at 4 °C in darkness for 45 min and washed twice with a PBS washing solution by centrifugation. Next, the cells were treated with 10 µL of goat anti-human IgG (FC)/FITC, incubated at 4 °C in darkness for 20 min, washed twice with a washing solution by centrifugation, and analyzed using flow cytometry (NovoCyte D3010).

Cell proliferation

T cells were washed and then resuspended in 100 mL of PBS at a concentration of up to 1 × 107 cells per mL. They were stained with 100 mL of 2.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE) (BioLegend, USA) to achieve a final concentration of 1.0 mM. This staining was performed for 20 min at 37 °C. The reaction was stopped by adding RPMI-1640 medium supplemented with 10% FBS, and the cells were washed twice. For the coculture assay, T cells were incubated with target cells at an effector-to-target (E:T) ratio of 1:1 for 48 h. The E:T ratio represents the ratio of the absolute number of CAR-T cells to target cells. The number of T cells used in the assay was the same as that of the 2G CAR-T group.

Cytotoxicity assay

In the experiment, CFSE-labeled target cells were cocultured with effector T cells at the specified ratios for either 12–16 h or 6–8 h. After the coculture period, the cells were harvested and Annexin V and 7-AAD were added for flow cytometric analysis. The remaining live target cells were identified as CFSE + Annexin V- 7-AAD-. The E:T ratios represented the ratios of the absolute number of CAR-T cells to target cells. The number of mock-T cells used in the experiment was the same as that in the 2G CAR-T group. All experiments were conducted in triplicate to ensure reliability and consistency. To calculate the cytotoxicity efficiency of CAR-T cells, the ratio of dead target cells to the total number of dead and live cells was determined. Dead cells were identified as Annexin V + 7-AAD- or Annexin V + 7-AAD + cells.

Cytokines production

To assess cytokine production, effector cells (5 × 104) and target cells (5 × 104) were cocultured at a 1:1 ratio in RPMI medium supplemented with 10% FBS and 10% human serum for 24 h. The concentration of cytokines in the culture supernatant and mouse serum was measured using enzyme-linked immunosorbent assay (ELISA) kits. Specifically, ELISA kits from MultiSciences Biotech Co., Ltd. (China) were used to measure the levels of IFN-γ, TNF-α, and IL-2. Additionally, a human premixed multi-analyte flow assay kit from BioLegend (USA) was used to simultaneously measure the concentration of a panel of soluble factors (IL-2, IL-6, IL-10, IL-21, IFN-γ, and TNF-α) in the same sample. The E:T ratio represented the ratio of the absolute number of CAR-T cells to target cells. The number of T cells used in the experiment was the same as that in the 2G CAR-T group.

Flow cytometry

Anti-human antibodies were obtained from Becton Dickinson, BioLegend, and Miltenyi Biotec. The Accuri C6, FACS Calibur, and BD FACSAria™ II cell sorter were used for analyzing various samples. Anti-human antibodies were purchased from BioLegend, eBioscience, Acrobiosystems, or BD. Cells were isolated from in vitro cultures or animals, washed with PBS supplemented with 2% FCS, and stained on ice after blocking Fc receptors. In all analyses, the population of interest was gated based on forward vs. side scatter characteristics, followed by singlet gating. The differentiation stage of T cells was labeled as naïve T (TN): CD45RA + CD45RO-, stem cell memory T (TSCM): CD45RA + CD45RO+, central memory T (TCM): CD45RA-CCR7+, and effector memory T (TEM): CD45RA-CCR7- [30].

Mouse xenograft tumor model

In the mouse xenograft tumor model, the animal experiments were conducted at the National Beijing Center for Drug Safety Evaluation and Research and at the SAFE Pharmaceutical Research Institute Co., Ltd. The study was conducted following the guidelines and regulations set by the Institutional Animal Care and Use Committee (IACUC-2019-001). Female NSG mice, aged 6–8 weeks, were used for the experiments. To establish the NALM-6-PDL1 acute precursor B-ALL models, 106 tumor cells were intravenously injected with PBS. Tumor growth was monitored by measuring the total bioluminescent flux using a Xenogen Imaging System (PerkinElmer-IVIS Lumina III). Peripheral blood samples were collected from the mice via the tail vein. Furthermore, different organs were dissected from the NSG mice, and cells from these organs were harvested. The expression of murine PDL1/2 in different tissues was detected. Due to the cross-species binding affinity of human PD1 to murine PDL1/2, human PDL1/2 antibodies were used to detect the expression of murine PDL1/2.

RNA-sequencing and bioinformatics analysis

Total RNA was purified from 2G CAR-T cells or PEPT/PE8T CAR-T cells after incubating with NALM-6 cells with a RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen). RNA integrity was verified with an Agilent TapeStation (RIN). The further preparation for sequencing and bioinformatics analysis was performed as previously described [31].

Analysis of metabolic parameters

Basal oxygen consumption rate (OCR) was measured, followed by serial additions of oligomycin (an inhibitor of ATP synthesis), carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (FCCP; an uncoupling ionophore), and rotenone with antimycin A (blocking agents for complexes I and III of the electron transport chain, respectively) to discern the relative contributions of mitochondrial and non-mitochondrial mechanism of oxygen consumption [49]. Mitochondrial function was assessed with an extracellular flux analyzer (Seahorse Bioscience). Individual wells of an XF96 cell culture microplates were coated with CellTak in accordance with the manufacturer’s instructions. The matrix was adsorbed overnight at 37 °C, aspirated, air-dried, and stored at 4 °C until use. Mitochondrial function was assessed on day 10 (incubated with NALM-6-PDL1 for 48 h). To assay mitochondrial function, we centrifuged T cells at 500 × g for 5 min. Cell pellets were resuspended in XF assay medium (non-buffered RPMI 1640) containing 5.5 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate and seeded at 5 × 104 cells per well. The microplate was centrifuged at 500 × g for 5 min and incubated in standard culture conditions for 60 min. During instrument calibration (30 min) the cells were switched to a CO2-free (37 °C) incubator. XF96 assay cartridges were calibrated in accordance with the manufacturer’s instructions. Cellular OCRs were measured under basal conditions and, following treatment with 1.5 mM oligomycin, 1.5 mM FCCP, and 40 nM rotenone, with 1mM antimycin A (XF Cell Mito Stress kit, Seahorse Bioscience).

Statistical analysis

Statistical analyses were performed using Prism version 7.0 (GraphPad). For studies comparing two groups, we utilized an unpair Students t-test. Survival data were analyzed by the log-rank test, and survival curves were assessed using the Kaplan-Meier method.