The following cell lines were used: K562 (a chronic myelogenous leukemia cell line, ATCC #CCL-243), Jurkat, clone E6-1 (an acute T cell leukemia cell line, ATCC #TIB-152), A549 (a lung cancer cell line, ATCC #CCL-185), NCI-H292 (a lung cancer cell line, ATCC #CRL-1848), SKOV3 (an ovarian cancer cell line, HTB-77) and HEK293T (an embryonic kidney cell line, ATCC #CRL-3216). All cell lines were purchased from ATCC and maintained in RPMI 1640 (Corning #10-040-CVR) supplemented with 10% fetal bovine serum (FBS) (BI #04-001-1acs) and 1% penicillin-streptomycin (Corning #30-002-CI), except for HEK293T cells, which were cultured in DMEM (Corning #10-013-CV) supplemented with 10% FBS and 1% penicillin-streptomycin. All tumor cell lines used in our study expressed little or no PD-L1 (Additional file 2: Figure S2). Thus, pseudotyped viruses carrying the PD-L1 gene were delivered into tumor cells to produce lines that had high, stable expression of PD-L1, e.g., K562-PD-L1, A549-PD-L1, NCI-H292-PD-L1 and SKOV3-PD-L1. Furthermore, the firefly luciferase gene or CD19 was also delivered into tumor cells via pseudotyped lentiviruses to generate A549-Luc, A549-Luc-PD-L1, A549-Luc-CD19, A549-Luc-CD19-PD-L1, NCI-H292-Luc-CD19, NCI-H292-Luc-CD19-PD-L1, SKOV3-Luc and SKOV3-Luc-PD-L1. These cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.
CAR and CCR designs
DNA encoding the human CD8 signal peptide (SP) (NP_001759.3 aa 1-21), human PD-L1-binding scFv, Flag tag (DYKDDDDK), the hinger spacer, transmembrane and signaling domain of human CD28 (NP_006130.1 aa 114-220), the human 4-1BB signaling domain (AAA53133.1 aa 209-255) and the human CD3ζ cytosolic domain (NP_932170 aa 52-164) was cloned into the empty lentiviral transfer plasmid (pHAGE_EF1α_MCS_IRES_ZsGreen) to generate the recombinant lentiviral transfer plasmid containing PD-L1 CAR (pHAGE_PD-L1-28BBz) (Additional file 1: Figure S1a). The expression cassette encoding human CD8 SP, anti-human CD19 scFv (FMC63 clone), human HER2 scFv (4D5 clone) or low-affinity HER2 scFv (4D5-5 clone), Myc tag (EQKLISEEDL), the hinger and transmembrane domain of CD8 (NP_001759.3 aa 138-206) and the CD3ζ cytosolic domain was cloned into another empty lentiviral transfer plasmid (pKL_EF1α_MCS_P2A_EGFP) to generate recombinant lentiviral transfer plasmids carrying CD19 CAR (pKL_CD19-z) or HER2 CAR (pKL_HER2-z, pKL_La-HER2-z) (Fig. 1a and Additional file 3: Figure S3). The artificial gene encoding both the CD19/HER2 CAR and the PD-L1 CCR is composed of the CD8 SP, PD-L1 scFv, Flag tag, and the hinger, transmembrane and signaling domain of CD28 (PD-L1-28). The CD19/HER2 CAR was linked to the PD-L1 CCR via the self-cleaving T2A peptide sequence. The expression cassette encoding both the CAR and CCR was also cloned into the empty pKL_EF1α_MCS_P2A_EGFP plasmid to generate the recombinant pKL_CD19-z-PD-L1-28 and pKL_HER2-z-PD-L1-28 plasmids (Fig. 1a and Additional file 3: Figure S3). All genes were synthesized by Generay Biotech (Shanghai) Co., Ltd. The lentiviral transfer plasmids also encoded enhanced green fluorescent protein (EGFP) or ZsGreen to evaluate the transduction efficiency of pseudoviruses carrying the CAR or CCR.
Transient lentiviral supernatant was produced as described below. Lentiviral vectors were prepared by transient transfection of HEK293T cells using TurboFect transfection reagent (Thermo Scientific #R0531). HEK293T cells (6×106) cultured in 10-cm tissue culture dishes were transfected with the lentiviral transfer plasmid (3 μg), the VSV-G envelope plasmid PMD2.G (Addgene #12259) (3 μg) and the packaging plasmid psPAX2 encoding gag-pol (Addgene #12260) (9 μg). The lentiviral supernatant was harvested 48 h after transfection and filtered through a 0.45-μm filter (PALL #4614). Lentiviral particles were further concentrated by ultracentrifugation for 2 h at 28000 rpm with a Beckman SW28 rotor (Beckman) before use.
T cells transduction and expansion
Frozen human peripheral blood mononuclear cells (PBMCs) were obtained from Shanghai Public Health Clinical Center. PBMCs were thawed in T cell growth medium (TCM), consisting of X-VIVO 15 medium (Lonza #BE02-060F), human IL-7 (R&D systems #P13232), human IL-15 (R&D systems #P40933) and human IL-21 (Novoprotein #GMP-CC45), and then rested for 4~6 h. The thawed PBMCs were further sorted into CD4+ and CD8+ T cells with an EasySep™ Human CD4+ T Cell Enrichment Kit (STEMCELLS #19052) and EasySep™ Human CD8+ T Cell Enrichment Kit (STEMCELLS #19053), respectively. Before transduction, PBMCs or sorted CD4+/CD8+ T cells were stimulated for 24~36 h with anti-hCD3 and anti-hCD28-coated immunobeads at a bead-to-cell ratio of 1:1 in TCM. The activated T cells were incubated with freshly concentrated lentiviral vectors in NovoNectin (Novoprotein # GMP-CH38)-coated 48-well flat plates at 32℃ and centrifuged at 1000×g for 1.5 h. The culture medium was changed to fresh TCM overnight. The immunobeads were removed 6~7 days post-transduction, and the T cells were expanded until they were rested and could be used in further assays. During ex vivo expansion, the TCM was replenished, and the cell density was adjusted to 0.5~2×106/mL every 3 days.
Generation of CAR-engineered Jurkat T cells
Jurkat T cells were transduced with lentiviral particles carrying PD-L1-28BBz, CD19-z or CD19-z-PD-L1-28. Jurkat T cells expressing EGFP/ZsGreen were sorted by flow cytometry (BD FACS Aria II). The sorted Jurkat T cells were expanded and then used in further assays.
Cell aggregation assay
To determine whether the PD-L1 CAR-engineered Jurkat T cells bound to PD-L1 presented on the tumor cell surface to promote cell aggregation, PD-L1-positive K562 tumor cells labeled with Cell Proliferation Dye eFluor 670 (Invitrogen #65-0840) were mixed with PD-L1 CAR-modified Jurkat T cells or untransduced Jurkat T cells labeled with CellTrace CFSE (Invitrogen #C34554) at an effector: target (E: T) ratio of 1:1 in a 1.5 mL eppendorf tube at room temperature (RT) for 1 h. The proportion of cells forming heterologous cell aggregates (eFluor 670+CFSE+) was assessed by flow cytometry (BD LSRFortessa).
Surface immunostaining and flow cytometry
For tumor cells expressing CD19 and/or PD-L1, 5×105 tumor cells were harvested and washed twice with FACS buffer (1× PBS containing 2% FBS). Then, tumor cells were stained with 0.5 μL of APC/Cy7-conjugated mouse anti-human CD19 (BD Pharmingen #557791) or 0.5 μL of APC-conjugated mouse anti-human PD-L1 (eBioscience #17-5983-73) at 4°C for 30 min, washed with FACS buffer twice, and resuspended in FACS buffer for assessment. Additionally, 5×105 washed tumor cells were incubated with 2 μg/mL of trastuzumab prepared in our laboratory at 4℃ for 30 min, washed twice with FACS buffer, and further stained with 0.5 μL of PE-conjugated anti-human IgG Fc (Biolegend #409304) at 4℃ for 30 min, washed with FACS buffer twice, and then resuspended in FACS buffer to detect HER2. To detect the HER2 CAR, PD-L1 CAR or PD-L1 CCR presented on the T cells surface, T cells were stained with Alexa Fluor 647-conjugated anti-Myc tag (CST #2233S) or PE-conjugated anti-DYKDDDDK (Biolegend #637310). T cells (5×105) were harvested and washed twice with FACS buffer. For Myc or Flag tag staining, T cells were stained with 0.5 μL of Alexa Fluor 647-conjugated anti-Myc tag or PE-anti-DYKDDDDK at 4°C for 30 min, washed twice with FACS buffer, and then resuspended in FACS buffer for detection. Fluorescence was assayed using a BD LSRFortessa, and all FACS data were analyzed with FlowJo V10 software.
In vitro stimulation of CAR-T cells and quantitation of cytokines
CAR-T cells (1×105) were co-cultured with K562 cells at an effector: target (E:T) ratio of 1:1. After mixing CAR-T cells and K562 cells in a 96-well round-bottom plate, the plate was centrifuged for 1 min at 400×g to force cell interactions. For adherent tumor cells, 1×105 CAR-T cells were co-cultured with tumor cells at an E:T ratio of 3:1 in a 96-well flat-bottom plate. The co-culture supernatant was collected after 24 h and stored at -20°C for further quantitation. IL-2, IFN-γ or TNF-α levels in the culture supernatant were determined using the Human IL-2 ELISA Set (BD Biosciences #555190), Human IFN-γ ELISA Set (BD Biosciences #555142) or BD CBA Human Th1/Th2/Th17 Cytokine Kit (BD Biosciences #560484).
In vitro evaluation of CAR-T cells cytotoxicity
Tumor cell lysis by CAR-T cells was assessed by using an 18-h luciferase-based killing assay. Briefly, tumor cells expressing target antigen and luciferase were plated in triplicate in a 96-well black flat-bottom plate (Greiner #655090) at a density of 1×104 cells per well and grown for 18~24 h. CAR-T cells were added to the plate at E:T ratios of 0.5:1, 1:1, 2:1 and 4:1 and then cultured for another 18 h. The culture supernatant was removed, and the viability of the tumor cells was assessed by quantifying the firefly luciferase fluorescence intensity with a GloMax® 96 reader (Promega #E6521) using a Luciferase Assay System (Promega #E1501). The formula used to calculate the percent normalized cytotoxicity is as follows:
100% - (luciferase fluorescence intensity in untransduced T cells well - luciferase fluorescence intensity in assay well)/ (luciferase fluorescence intensity in untransduced T cells well).
A flow cytometry-based assay was also used to measure cytotoxicity for CAR-T cells co-cultured with multiple cell lines (e.g. K562-PD-L1, A549, NCI-H292, and SKOV3). Briefly, CAR-T cells were co-incubated at different ratios (e.g. 1:1, 2:1 and 4:1) with pre-labeled adherent tumor cells (e.g. A549, NCI-H292, and SKOV3) or K562-PD-L1 cells. The adherent tumor cells were labeled with Cell Proliferation Dye eFluor 450 (Invitrogen #65-0842-85), and K562-PD-L1 cells were labeled with Cell Proliferation Dye eFluor 670 at a final concentration of 5 μM to allow their discrimination from CAR-T cells. After 12h, dead target cells were identified by propidium iodide (PI) staining (BD #556547) using flow cytometry. The formula used to calculate the percent normalized cytotoxicity is as follows:
100% - (100% - % of dead cells in assay well)/ (100% - % of dead cells in untransduced T cells well).
Detection of the in vitro proliferation of CAR-T cells
For in vitro proliferation assays, single-targeted and dual-targeted CAR-T cells were washed with 1× PBS and then labeled with Cell Proliferation Dye eFluor 670 at a final concentration of 5 μM, according to the manufacturer’s instructions. Tumor cells expressing CD19/HER2 or PD-L1 were treated with 50 μg/mL mitomycin C (Biotech well #WF0197) to result in replication-defective tumor cells. Then, these pre-labeled CAR-T cells were co-cultured with pretreated tumor cells at an E:T ratio of 2:1 in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin, and the mixed cells were collected for flow cytometry analysis. Finally, the proliferation of CAR-T cells was assayed by monitoring the dilution of the cell proliferation dye after four days of co-culture.
Xenograft dual-tumor models
The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Public Health Clinical Center. Female NOD-Prkdcscid Il2rgtm1/Bcgen (B-NDG) (Biocytogen) mice aged 6~8 weeks were subcutaneously inoculated with four xenograft tumors in the right flank: 1) 5×106 CD19+ and 5×106 CD19+PD-L1+ A549 cells; 2) 2×106 CD19+ and 2×106 CD19+PD-L1+ NCI-H292 cells; 3) 2×106 HER2+ and 2×106 HER2+PD-L1+ NCI-H292 tumor cells; or 4) 1×106 HER2+ and 1×106 HER2+PD-L1+ SKOV3 cells. Five or ten days after tumor inoculation, 2×106 CD4+ and CD8+ T cells were injected intravenously into the A549 tumor-bearing mice, while 4×106 CD4+ and CD8+ T cells were injected into the NCI-H292/SKOV3 tumor-bearing mice. These T cells were either untransduced or engineered with single-targeted CAR or dual-targeted CAR. Tumor size was monitored by calipers every 3, 5 or 10 days after T cells transfer, and tumor volume was calculated with the following formula: V = (length × width2)/2. Mice were considered dead when the tumor size reached the euthanasia criteria.
All data are presented as the mean ± standard error of the mean (SEM) unless otherwise described. Statistical differences were determined by a paired Student’s t-test (two-tailed) unless otherwise noted. The statistical significance of differences among three or more groups was analyzed by one-way ANOVA with Tukey’s test for further multiple comparisons. A P-value < 0.05 was considered statistically significant. All statistical analyses were performed with Prism 7.0 (GraphPad), and statistical significance was reported as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.