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Flow cytometry enables the simultaneous detection of multiple surface and intracellular antigens for proteomic profiling of cells. This allows characterization and identification of specific cell subtypes within a heterogeneous population and is usually called immunophenotyping. Antigen-specific antibodies, conjugated to various fluorophores, are incubated with the sample to identify each marker. Fluorescent light of various wavelengths can be separated, detected, and converted into a digital signal in a flow cytometer. Here we describe an eight-color experiment to identify key peripheral blood cell types; however, this technique can be expanded to detect more than 30 parameters simultaneously.

Flow cytometry enables the simultaneous detection of multiple surface and intracellular antigens for proteomic profiling of cells. This allows characterization and identification of specific cell subtypes within a heterogeneous population and is usually called immunophenotyping. Antigen-specific antibodies, conjugated to various fluorophores, are incubated with the sample to identify each marker. Fluorescent light of various wavelengths can be separated, detected, and converted into a digital signal in a flow cytometer. Here we describe an eight-color experiment to identify key peripheral blood cell types; however, this technique can be expanded to detect more than 30 parameters simultaneously.

Background aims Natural killer (NK) cells have the potential to become a successful immunotherapy as they can target malignant cells without being direct effectors of graft-versus-host disease. Our group has previously shown that large numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34 + cells. To produce a clinically relevant and effective immunotherapy, we hypothesized that it is essential that the NK cells are able to proliferate and persist in vivo while maintaining an optimal activation status and killing capacity. Methods We evaluated the proliferation capacity, telomere length and terminal differentiation markers expressed by NK cells differentiated in vitro . We also determined how their cytotoxicity compared with peripheral blood (PB) NK cells and CBNK cells when targeting patient acute myeloid leukemia (AML) blasts and solid tumor cell lines. Results We found that the differentiated NK cells could respond to interleukin-2 and proliferate in vitro. Telomere length was significantly increased, whereas CD57 expression was significantly reduced compared with PBNK cells. The cytotoxicity of the differentiated NK cells was equivalent to that of the PBNK and CBNK cell controls, and priming consistently led to higher levels of killing of patient leukemic blasts and solid tumor cell lines in vitro . Interestingly, this activation step was not required to observe killing of patient AML blasts in vivo. Conclusion We are able to generate NK cells from CBCD34 + cells in high numbers, allowing for multiple infusions of highly cytotoxic NK cells that have potential to further proliferate in vivo , making them a desirable product for application as an immunotherapy in the clinic.

Standing et al. report a novel autoinflammatory disease caused by a homozygous missense mutation in the actin-regulating protein WDR1. The disease is characterized by periodic fevers, immunodeficiency, and thrombocytopenia, with increased polymerized actin in immune cells and increased IL-18 secretion., The importance of actin dynamics in the activation of the inflammasome is becoming increasingly apparent. IL-1β, which is activated by the inflammasome, is known to be central to the pathogenesis of many monogenic autoinflammatory diseases. However, evidence from an autoinflammatory murine model indicates that IL-18, the other cytokine triggered by inflammasome activity, is important in its own right. In this model, autoinflammation was caused by mutation in the actin regulatory gene WDR1. We report a homozygous missense mutation in WDR1 in two siblings causing periodic fevers with immunodeficiency and thrombocytopenia. We found impaired actin dynamics in patient immune cells. Patients had high serum levels of IL-18, without a corresponding increase in IL-18–binding protein or IL-1β, and their cells also secreted more IL-18 but not IL-1β in culture. We found increased caspase-1 cleavage within patient monocytes indicative of increased inflammasome activity. We transfected HEK293T cells with pyrin and wild-type and mutated WDR1. Mutant protein formed aggregates that appeared to accumulate pyrin; this could potentially precipitate inflammasome assembly. We have extended the findings from the mouse model to highlight the importance of WDR1 and actin regulation in the activation of the inflammasome, and in human autoinflammation.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Background Mutations in the perforin 1 (PRF1) gene account for up to 58% of familial hemophagocytic lymphohistiocytosis syndromes. The resulting defects in effector cell cytotoxicity lead to hypercytokinemia and hyperactivation with inflammation in various organs. Objective We sought to determine whether autologous gene-corrected T cells can restore cytotoxic function, reduce disease activity, and prevent hemophagocytic lymphohistiocytosis (HLH) symptoms in in vivo models. Methods We developed a gammaretroviral vector to transduce murine CD8 T cells in the Prf−/− mouse model. To verify functional correction of Prf−/− CD8 T cells in vivo, we used a lymphocytic choriomeningitis virus (LCMV) epitope–transfected murine lung carcinoma cell tumor model. Furthermore, we challenged gene-corrected and uncorrected mice with LCMV. One patient sample was transduced with a PRF1-encoding lentiviral vector to study restoration of cytotoxicity in human cells. Results We demonstrated efficient engraftment and functional reconstitution of cytotoxicity after intravenous administration of gene-corrected Prf−/− CD8 T cells into Prf−/− mice. In the tumor model infusion of Prf−/− gene–corrected CD8 T cells eliminated the tumor as efficiently as transplantation of wild-type CD8 T cells. Similarly, mice reconstituted with gene-corrected Prf−/− CD8 T cells displayed complete protection from the HLH phenotype after infection with LCMV. Patients' cells showed correction of cytotoxicity in human CD8 T cells after transduction. Conclusion These data demonstrate the potential application of T-cell gene therapy in reconstituting cytotoxic function and protection against HLH in the setting of perforin deficiency.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.

The actin cytoskeleton is crucial at many junctures of normal immune function, and consequently there are many immune specific regulators of actin dynamics. A growing number of primary immunodeficiencies are being defined as caused by mutations in the genes encoding these regulators. In addition to immunodeficiency, immune dysregulation and autoinflammation are increasingly recognised to arise from defects within this pathway.

Introduction Human hematopoietic stem cell transplantation (HSCT) has been used to treat a range of hematological and immunological disorders. As a result, the demand for hematopoietic stem cells (HSC) in clinical applications is increasing. Amniotic fluid stem cells (AFSC) serve as a potential alternative cell source for therapy. Amniotic fluid can be derived by amniocentesis or therapeutic amniodrainage. AFSC are multi-potent, have low risk of tumorigenicity, can be expanded and do not have legal or ethical limitations. The significant hematopoietic activity of murine AFSC led us to explore the potential of human AFSC (CD117/c-Kit+) towards hematopoietic differentiation and to reconstitution in vivo. Methods Human AFSC (2nd and 3rd trimester) and cord blood HSC (CB-HSC; control) were selected for CD117 and CD34 respectively using a MoFlo XDP sorter. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NOD-SCID/IL2rγnull (NSG) mice (n=6/group). Hematopoietic engraftment of human cells (% of human CD45+ within total CD45+) and multi-lineage reconstitution (myeloid: CD13, CD14, CD15 and lymphoid: CD3, CD4 and CD8) were assessed at 16 weeks in blood, bone marrow (BM) and spleen by flow cytometry. For subsequent secondary transplants, BM mononuclear cells (MNC) derived from BM harvested from primary recipients of mice were intravenously injected into secondary recipients (1.5x107 MNC in 200μl PBS). Hematopoietic engraftment was assessed at 16 weeks post-transplantation (n=6/group). For further analysis of human donor cell engraftment, Q-PCR was performed on spleen samples harvested from primary and secondary recipients using oligonucleotide primers specific for human ALU repeat sequences; Immunohistochemistry was carried out using anti-human CD45 antibody and detected with a commercially available kit (Dako EnVision Plus, Dako). Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results Human AFSC engrafted the hematopoietic system of NSG mice at levels similar to the ones achieved with CB-HSC (blood: AFSC 7.5±1.3% vs. CB-HSC 6.1±2.2%, p=0.6; BM: AFSC 46.3±7.9% vs. CB-HSC 38.3±8.2%, p=0.6; spleen: AFSC 39.6±9.3% vs. CB-HSC 34.7±10.5%, p=0.7). Similarly, at 16 weeks following secondary transplantation, human donor cell engraftment was comparable between groups in blood (AFSC 11.5 ± 3.9% vs. CB-HSC 16.9 ± 3.9%, p=0.3) and other hematopoietic tissues. Q-PCR and immunohistochemistry confirmed donor cell engraftment in AFSC and CB-HSC groups. Importantly, there were no differences between groups in multi-lineage differentiation at 16 weeks post primary and secondary transplantation. Conclusion Human CD117/c-Kit+ AFSC have functional, multi-lineage hematopoietic potential that is similar to the current "gold-standard" stem cell source for hematopoietic transplantation. The ease of isolation during early gestation, as well as their gene-engineering and expansion potential make human AFSC a novel autologous fetal cell source for pre- and post-natal therapy of inherited hematological disorders. Disclosures No relevant conflicts of interest to declare.