CD19-specific chimeric antigen receptor (CAR19) T-cells effectively induce remission of B-cell malignancy, but the cost and complexity of production using viral vectors is a factor limiting widespread application. Furthermore, the small cargo capacity of viral vectors may hamper future development of more heavily engineered CAR T-cells. We demonstrated the feasibility of generating CAR19 T-cells from HLA-matched donors of sibling allogeneic hematopoietic stem cell transplant (HSCT) patients via a simple and inexpensive method using the high-capacity piggyBac transposon. A cohort of 10 patients with relapsed or refractory B-cell acute lymphoblastic leukemia or aggressive lymphoma following HSCT were the first human subjects to receive piggyBac-generated CAR19 T-cells. Treatment with intra-patient escalating doses of CAR19 T-cells was effective, with all 9 evaluable patients achieving complete remission. At a median follow-up of 18.0 months, 5 patients remained in complete remission of B-cell malignancy. One patient died of viral sepsis. Four patients developed cytokine release syndrome of maximum grade 2, and no neurotoxicity or new graft-versus-host disease occurred. However, two patients developed malignant CAR19 T-cell tumors, one of whom was successfully treated; one patient died of the secondary tumor. The piggyBac system represents a feasible alternative to viral vectors for the generation of effective CAR19 T-cells, but its oncogenic potential in the context of the described production process will need to be addressed before any further clinical use is possible. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Flow cytometry detection of neutrophil NETs markers is feasible in whole blood and correlates with serum markers of NETs. We propose the flow cytometry detection of MPO/H3cit positive neutrophils and serum dsDNA as simple methods to quantify cellular and extracellular NET markers in patients with thrombosis and sepsis.

There are three known splice variants of Type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPkin Iγ): PIPkins Iγ87, Iγ90, and the most recently cloned (Giudici, M.L., Emson, P.C. and Irvine, R.F. (2004) A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma. Biochem. J. 379, 489–496) PIPkin IγC (here called PIPkin Iγ93). Here, we have explored the subcellular localisation and mobility of Type I PIPkins in transfected cells by confocal microscopy and flourescence recovery after photobleaching. The unique behaviour shown by PIPkin Iγ93 is consistent with its suggested distinct function. Moreover, the markedly different localisation and mobility of active versus inactive PIPkin Iγ93 provide insights into the factors that dictate cellular targeting of Type Iγ PIPkins.

Objective. Adoptive immunotherapy with ex vivo expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy. Methods. Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137 neg fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures. Results. PRAME-stimulated cultures (n = 10) had mean expansion of 2500-fold at day 18. Mean CD3 + percentage was 96% with CD4: CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-c (mean: 12.2%) and TNF-a (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype