Proliferation of T cells was analyzed by flow cytometry

Proliferation of T cells was analyzed by flow cytometry. CAR T cell cytotoxicity assay was performed as described (20) with some modifications. human anti-Siglec-6 antibody (JML1), which was identified in a ERK CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels and and in a xenograft mouse model. Methods Human blood samples and cells Healthy blood from anonymous donors were obtained with written consent from the Department of Transfusion Medicine, NIH Clinical Center, and analyzed with the approval of the ethical review committee of the NIH. Dutogliptin CLL patient samples were obtained after written informed consent in accordance with the Declaration of Helsinki, applicable federal regulations, and requirements from the National Heart, Lung, and Blood Institute Institutional Review Board. The clinical study is registered at as “type”:”clinical-trial”,”attrs”:”text”:”NCT00923507″,”term_id”:”NCT00923507″NCT00923507 and “type”:”clinical-trial”,”attrs”:”text”:”NCT00071045″,”term_id”:”NCT00071045″NCT00071045. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Ficoll-Hypaque (GE Healthcare Biosciences, Pittsburgh, PA) by gradient centrifugation. The following cell lines were cultured in complete media and used as a target in assays: CCRF-CEM, MEC1, Nalm6 and U937. MEC1-001 and MEC1-002 were descripted previously (17), and were tested unfavorable for mycoplasma contamination. Both B- and T- cells were isolated from buffy coat using human CD19 microbeads and pan T cell isolation kit (Miltenyi Biotech, Auburn, CA). Human CD34+ hematopoietic stem cells (HSCs) were purchased from StemCell Technologies (Vancouver, BC, Canada). MEC1-001-Siglec-6 Transgenic (TG) cells we generated by retroviral transduction with pEV-Thy1.1-hSiglec-6. The CD19-CAR construct corresponds to the previously described MSGV-FMC63-28Z (19). RNA preparation and quantitative real-time PCR Total RNA was extracted from enriched B-, T- cells and immortalized cell lines using RNeasy Miniprep kit (Qiagen, Dutogliptin Valencia, CA) according to the manufacturers instructions. Complementary DNA (cDNA) was synthesized with High-Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA) using 1 g of total RNA in a 20-l reaction volume, one-hundredth of which was used as a template for real-time PCR reaction in a QuantStudio 3 Real-Time PCR system (Applied Biosystems) using the Taqman probe (Applied Biosystems) according to the manufacturers instructions. Human normal tissue cDNA array was purchased from OriGene Technologies (Rockville, MD). Each PCR procedure included a non-template unfavorable control reaction. The level of 18s rRNA expression was used as the internal control, and C(t) values were calculated according to the 2?C(t) method. Each sample subjected to quantitative real-time PCR was analyzed in triplicate and repeated 3 times. Immunohistochemistry and tissue arrays Immunohistochemistry (IHC) analysis was performed using IHC protocols Dutogliptin on commercial bone marrow, lymphoma, normal lymph node and normal tissue Dutogliptin arrays BM241, LM721 and BCN921 US Biomax, Rockville, MD) using rabbit anti-human Siglec-6 polyclonal antibodies (Ab38581, Abcam, Cambridge, MA). Generation of the Siglec 6-specific JML1 CAR Fragments corresponding to JML1-CAR scFv constructs were generated as JML1VL-linker-JML1VH (linker amino acid sequence= GSTSGSGKPGSGEGSTKG) and cloned onto the CD19-CAR MSGV FMC63-28Z retroviral vector by replacing the fragments including CD19 scFv via XhoI and BmgBI restriction enzymes and T4 ligation. The resulting constructs contained a JML1 scFv plus a longer hinge region derived from CD28 (amino acid sequence= ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFACD) or a shorter hinge region derived from IgG4 (amino acid sequence= ESKYGPPCPPCP), followed by the CD28 transmembrane and co-stimulatory domains and by the Dutogliptin CD3 zeta stimulatory cytoplastic domain name. Retrovirus was produced using a 293GP packaging cell line by transient co-transfection with retroviral vector plasmid (JML1 CAR and CD19 CAR construct) and a plasmid encoding the RD114 envelope protein. Culture supernatant made up of retroviral particles was harvested after 48-72hs and stored at ?80C. To prepare CAR transduced T cells, cells were prestimulated with an anti-CD3 monoclonal antibody (clone OKT-3, 50 ng/mL) for 48 hours and centrifuged at 32C for 2 hours with viral particles onto retronectin (10 g/mL, Takara Bio, Mountain View, CA)-coated multi-well plates. After transduction, cells were expanded for 3-4 weeks in AIM-V media containing human AB serum.

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