Generation of CD38-CAR T cells
Peripheral blood mononuclear cells (PBMCs) obtained from de-identified healthy donors at Gulf Coast Regional Blood Center (Houston, TX) were isolated from leukapheresis products by Ficoll-Paque (Cytiva, Marlborough, MA) gradient centrifugation. T cells were then negatively selected by the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada). These cells were activated by anti-CD3/CD28-coated beads (Life Technologies, Carlsbad, CA) at a cell-to-bead ratio of 1:3 with 200 U/ml of IL-2 (PeproTech, London, UK) in CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies). After 24–48 h of activation, cells were transduced by the addition of high-titer lentiviral particles expressing CD38-CAR, CD19-CAR, or green fluorescent protein (GFP). Transduced T cells were maintained at a concentration of 0.7 × 106 cells/ml for 7–10 days by cell enumeration every 2–3 days. Finally, the T cells were induced to proliferate using a previously described rapid expansion protocol (REP) .
Cell lines and reagents
All cell lines used in this study were listed in Supplemental Table 1. The MM cell lines (MM.1S, OPM-1, NCI-H929, RPMI-8226, KMS-12, and ANBL-6), Jurkat (T-ALL), and K562 (CML, the only myeloid cancer cell line) were purchased from the American Type Culture Collection (Manassas, VA). The Waldenstrom macroglobulinemia (WM) cell line RPCI-WM1 was obtained previously . NKTCL cell lines KHYG-1, HANK-1, and SNK-6 were gifts from Dr. Javeed Iqbal, College of Medicine, University of Nebraska Medical Center. NKTCL cell lines YT and NK-YS were gifts from Dr. John Chan, Department of Pathology, City of Hope National Medical Center. The NKTCL, MCL, and MM cell lines NK-92, JeKo-1, Granta-519, Mino, SP-53, and MM.1R were gifts from Dr. Qing Yi, Center for Translational Research in Hematological Malignancies, Houston Methodist Hospital. The DLBCL cell line WSU (WSU-DLCL) and WILL-2 were gifts from Dr. Ken H. Young, Department of Pathology, Duke University Medical Center. MOLT-4 was obtained from Molecular and Cellular Biology Tissue Culture Core Laboratory, Baylor College of Medicine. Cells were cultured in their respective media with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. NK-92 cell lines were cultured with α-MEM with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, and 100 U/ml recombinant IL-2. KHYG-1, HANK-1, NK-YS, and SNK-6 were cultured with 20 U/ml, 10 U/ml, 100 U/ml, and 300 U/ml recombinant IL-2, respectively. All cell lines were cultured at 37˚C with 5% CO2 under humidified conditions. Chemical reagents dimethyl sulfoxide and retinoic acid (cat. # R2625) were purchased from Sigma-Aldrich (St. Louis, MO). U73122 (cat. # S8011) and selumetinib (cat. # S1008) were obtained from Selleck Chemicals (Houston, TX). Ipatasertib (cat. #A3006), PF-431396 (cat. #A8692), and saracatinib (cat. #A2133) were obtained from ApexBio (Houston, TX). Proteome Profiler Human Phospho-Kinase Array Kit (cat. #ARY003C) was obtained from R & D Systems (Minneapolis, MN). Antibodies against β-actin (cat. #A5316) were obtained from Sigma-Aldrich. Anti-CD38 (cat. # ab108403) antibody was obtained from Abcam (Cambridge, UK). Anti-PYK2 (cat. #3292S) and anti-p-PYK2 (cat. #3291S) were obtained from Cell Signaling Technology (Danvers, MA).
Six to eight-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained at Baylor College of Medicine Animal Facility. All procedures were carried out with IACUC approval at Baylor College of Medicine. For the MM model, 1 × 106 firefly luciferase (fLuc)-expressing OPM-1 was intravenously injected into NSG mice. One week later, a single injection of 5 × 106 of CD38-CAR T cells or non-transduced T cells were injected into NSG mice through the tail vein. For the MCL and WM model, 0.8 × 106 fLuc-JeKo-1 or fLuc-RPCI-WM1 was injected into NSG mice, which were then treated with 5 × 106 of CD38-CAR T cells after 1 week. For the T-ALL model, 5 × 106 fLuc-MOLT-4 was intravenously injected into NSG mice, which were treated with a single injection of 8 × 106 of CD38-CAR T cells. For the therapeutic NKTCL model, 5 × 106 YT was subcutaneously injected into NSG mice, which were treated with two injections of 8 × 106 of CD38-CAR T cells at day 18 and 20. For the prophylactic NKTL model, 5 × 106 YT was subcutaneously injected into NSG mice, which were treated with a single injection of 5 × 106 of CD38-CAR T cells on day 10. For the SP-53-CAR T model, 3 × 106 SP-53 was subcutaneously injected into NSG mice, which were treated with two doses of 8 × 106 of CD38-CAR T cells after 5 days (with or without intraperitoneal ATRA treatment). For the SP-53-daratumumab model, 3 × 106 SP-53 was subcutaneously injected into NSG mice, which were treated with two doses of 1 × 107 of PBMCs (NK-cell-enriched) after 8 days (with ATRA and/or daratumumab) . Tumor burden was monitored either with a vernier caliper every 2–3 days or using an IVIS Imaging System (Caliper Life Sciences, Waltham, MA) that recorded bioluminescence from mice injected intraperitoneally with 150 mg/kg of D-luciferin (Gold Technology, St. Louis, MO) at indicated time points. Tumor volume was calculated according to the formula: tumor volume (mm3) = (length × width × width/2), where length is the longest diameter and width is the shortest. Living Image software (PerkinElmer, Waltham, MA) was used to visualize and calculate total luminescence.
Western blot analysis
Cells were lysed by 1 × RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA) with 1% SDS and protease and phosphatase inhibitors (Thermo Fisher Scientific) and then collected and centrifuged at 12,000 rpm for 15 min in 4˚C. The supernatant was measured with the BCA protein assay reagent (Thermo Fisher Scientific). Lysates were denatured in Laemmli sample buffer (Bio-Rad, Hercules, CA) and resolved by Tris–glycine SDS-PAGE (4–20% polyacrylamide, Mini-PROTEAN Precast Gels, Bio-Rad). After transferring to the polyvinyl difluoride membrane, the membrane was blocked with 5% non-fat dry milk in 0.1% TBS-Tween-20 for 2 h and incubated with the primary antibody at 4˚C overnight. HRP-conjugated anti-rabbit or anti-mouse IgG (Cell Signaling Technology) was used as the secondary antibody. Immunoreactive protein was visualized with the enhanced chemiluminescent (ECL) western blotting substrate (Thermo Fisher Scientific).
RNA preparation and qRT-PCR
Total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse-transcribed into cDNA with the iScript™ cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad). Gene expression levels were quantified by qRT-PCR performed on a QuantStudio 7 Pro qRT-PCR system. The qRT-PCR analysis was performed using primers for each gene, and the results were normalized to Beta-2-microglobulin (β2M) transcript levels. The difference in fold expression was calculated using the ΔΔCT method. Primers used against each gene were validated for specificity using BLAST and melting curve analysis.
Luciferase-based cytotoxicity assay in vitro
To measure cytotoxicity, 2–5 × 104 cells (MM.1S, OPM-1, HANK-1, MOLT-4, RPCI-WM1, JeKo-1, Granta-519, K562) expressing fLuc were co-cultured with T cells in a round-bottom 96-well plate (Corning, Corning, NY) at an indicated effector: target (E:T) ratio for 4 or 20 h at 37 °C. D-Luciferin (Thermo Fisher Scientific) was then added, and luminescence was measured with a Synergy HTX Multi-Mode Reader (BioTek Instruments, Winooski, VT). The lysis of tumor cells was calculated as [1-(target cells with CD38-CAR T cells/target cells with control T cells)] × 100%. Assays were performed in triplicate.
Anti-human CD3 (cat. #300308), CD28 (cat. #302907), CD38 (cat. #303526), CD95 (cat. #305607), PD-1 (cat. #329937), PD-L1 (cat. #329708), CTLA-4 (cat. #349906), CCR7 (cat. #353210), and CD45RA (cat. #304154) from BioLegend, San Diego, CA, and TIM-3 (cat. #17–3109-42) and CD107a (cat. #A15729) from Thermo Fisher Scientific were used to stain cells. Biotin-Protein L (cat. # M00097, GenScript, Piscataway, NJ) was used for CAR detection . CellTrace™ Far Red Cell Proliferation Kit (Thermo Fisher Scientific) was used to monitor T cell proliferation. All flow cytometry data were obtained in BD Accuri™ C6 Plus, BD LSRFortessa (BD Biosciences, San Jose, CA), or CyTek™ NL-3000 (Cytek Biosciences, Fremont, CA) and analyzed with FlowJo software (FlowJo).
Cytokine release in vitro
T cells and tumor cells were incubated at a 2:1 ratio for 20 h. Supernatants were harvested and subjected to ELISA for cytokine production according to the manufacturer’s instructions (R & D System).
Histology immunohistochemistry analyses (IHC)
Tissue samples were subjected to hematoxylin and eosin (H&E) staining or IHC staining by Human Tissue Acquisition and Pathology (HTAP) Core Lab in the Baylor College of Medicine following the manufacturer’s protocol, as described previously .
All statistical analyses were performed using the Prism v7.0 program (GraphPad Software, San Diego, CA). Data are presented as means ± standard deviation (SD). Comparisons between two groups were analyzed using the Student’s t-test, whereas that for three or more groups were tested using the two-way repeated-measures analysis of variance. Kaplan–Meier analysis and the log-rank test (Mantel-Cox) were used for survival analysis. A probability value of p ≤ 0.05 was considered statistically significant.