Each symbol represents an unbiased donor (n=5)

Each symbol represents an unbiased donor (n=5). NK-cell activation through CD28H. As 5,6-Dihydrouridine B7H7 is usually broadly expressed in tumor tissues, we engineered a CD28H chimeric antigen receptor (CD28H-CAR) consisting of full-length CD28H fused to the cytoplasmic domain name of T cell receptor chain. Remarkably, expression of CD28H-CAR in NK cells brought on lysis of B7H7+ HLA-E+ tumor cells by overriding inhibition by the HLA-E receptor NKG2A. The cytoplasmic domains of CD28H and of the chain were both required for this activity. Thus, CD28H is a powerful activation receptor of NK cells that broadens their antitumor activity and holds promise as a component of NK-based CARs for cancer immunotherapy. antitumor activity of the CD28H-CAR showed promising therapeutic potential. Materials and Methods Plasmids A plasmid made up of B7H7 cDNA was obtained from Harvard PlasmID Database (#HsCD00044662). B7H7 cDNA was amplified and cloned into the EcoRI and NotI cloning sites of pAc5.1/V5-His A vector (Thermo Fisher Scientific) for expression in S2 cells, and the EcoRI and NotI cloning sites of pCDH-EF1-T2A-Puro vector (System Biosciences) for expression in human cell lines. The cDNA of CD28H was obtained from Harvard PlasmID Database (#HsCD00416184) in the vector pLX304. CD28H cDNA was amplified and cloned into the EcoRI and NotI cloning sites of SAPK3 pCDH-EF1-T2A-Puro lentivirus vector (System Biosciences) for transduction of human cell lines. CD28H mutants and chimeras were generated using the In-Fusion HD cloning kit (Clontech) and verified by sequencing. All of the cDNAs cloned into the PCDH vector were in frame with the 2A-peptide. Expressed proteins could be detected by anti-2A antibody in immunoblots. All plasmid constructions were carried out using the In-Fusion HD cloning kit (Clontech). Cells Human NK cells were isolated from peripheral blood of healthy U.S. donors by unfavorable selection (STEMCELL Technologies). NK cells were resuspended in Iscoves modified Dulbeccos medium (IMDM; Gibco) 5,6-Dihydrouridine supplemented with 10% human serum (Valley Biomedical) and used within 4 days. IL2 and PHA activated 5,6-Dihydrouridine NK cells were cultured as described previously (18). Briefly, freshly isolated NK cells were cultured with irradiated autologous feeder cells in OpTimizer (Invitrogen) supplemented with 10% purified IL2 (Hemagen), 100 units/ml recombinant IL2 (Roche) and 5 g/ml phytohemagglutinin (PHA, Sigma), and expanded in the same medium without PHA and feeder cells. CD28H expression was tested after 2 weeks of activation. To obtain NK cells activated by NKp46 and CD2 plus IL2, freshly isolated NK cells were cultured in plates coated with 5 g/ml CD2 and NKp46 mAbs, in the presence of 100 units/ml recombinant IL2 (Roche). CD28H expression was tested at day 3, day 5, and day 7. NKL cells (obtained from M. J. Robertson, Indiana University Cancer Research Institute, Indianapolis, IN) and KHYG-1 cells were cultured in IMDM Medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco), 2 mM L-Glutamine (Gibco), and 100 units/ml recombinant IL-2 (Roche). 5,6-Dihydrouridine 721.221 cells (referred to as 221 cells), P815 cells (obtained from American Type Culture Collection, Manassas, VA), Daudi cells (ATCC Manassas, VA) and HDLM-2 cells (19) (obtained from T. Waldmann, NCI, NIH) were cultured in RPMI 1640 medium (Gibco) made up of 10% heat-inactivated fetal calf serum (Gibco) and 2 mM L-Glutamine (Gibco). 221 cells transfected with HLA-E (221.AEH), which included the HLA-A signal peptide to achieve proper HLA-E expression (20), were a gift from D. Geraghty (Fred Hutchinson Cancer Research Center, Seattle). Lenti-X 293T cells (Clontech) were cultured in DMEM medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 2 mM L-Glutamine (Gibco). Cells were mycoplasma-free, as tested by the NIH Office of Research Services. All cell lines used were maintained in culture for no longer than 2 months after thawing, and were authenticated by morphology, growth characteristics, expression of surface markers, and functional assays. Transfection and lentivirus production For S2 cells transfection, cells were transfected with plasmids for CD48 and B7H7 expression, both together or each one alone, together with a pAc5.1/V5-His A-puro plasmid for selection in 6 g/ml puromycin at 1/10th the amount of the expression plasmids. Resistant cells were cloned, and tested for CD48 and B7H7 expression. For production of lentivirus,.