David Darling

Protein transduction: an alternative to genetic intervention?

Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.

Eliciting cytotoxic T lymphocytes against acute myeloid leukemia-derived antigens: evaluation of dendritic cell-leukemia cell hybrids and other antigen-loading strategies for dendritic cell-based vaccination

Abstract. Dendritic cells (DC) have been successfully used in clinical pilot studies to induce tumor-specific immunity as well as clinical response in selected patients. However, DC-based immunotherapy remains a challenge and several parameters need to be examined in order to optimize the induction of anti-tumor immune responses. This study focuses on DC vaccination for leukemia and evaluates the in vitro efficacy of three different strategies for generating antigen-loaded DC-based vaccines for the induction of major histocompatibility complex (MHC) class I-restricted anti-leukemia cytotoxic T lymphocyte (CTL) responses. These included direct fusion of DC with leukemia cells to generate DC–leukemia cell hybrids, and DC pulsed with either apoptotic leukemia cell fragments or whole tumor cell lysates. Using either the U937 cell line or primary human acute myeloid leukemia blasts (AML), DC–leukemia cell hybrids were found to be the most potent in vitro inducers of CTL activity. DC pulsed with apoptotic tumor cell fragments were less efficient, but induced a more potent CTL response compared to tumor lysate-pulsed DC. The CTL responses were both MHC class I-restricted and antigen-specific, as shown by the inability of the CTL to lyse other control targets. The data presented here suggest that the method of antigen loading onto DC may be critical in the design of tumor vaccines.

Enhanced immune costimulatory activity of primary acute myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1

Gene modification of malignant cells to express immune stimulators (cytokines and immune costimulators) has provided the basis for a novel form of immunotherapy. Using a MPSV-based retroviral vector with hygromycin resistance gene as a selectable marker, we have studied retrovirus-mediated gene transfer of an immune costimulator, B7.1, into primary human acute myeloid leukaemia (AML) cells and the subsequent induction of immune costimulatory function. AML blasts from 10 patients were transduced by co-culture for 48 h with or without haemopoietic growth factors (HGFs). In the absence of HGFs, transduction efficiency (TE), as judged by % B7.1 expressing cells, was low, varying from 0.3 to 8.2% (median 1.5%). Addition of HGFs increased the median TE 1.8-fold with stem cell factor alone and 2.6-fold with SCF, interleukin-3 and GM-CSF. Effects on cell cycling alone could not explain this difference, suggesting other factors such as virus binding and promoter activity, are also involved. CFU-AL assays indicated a higher transduction efficiency of clonogenic cells, which was not improved by growth factors. Limited duration of cell growth prevented significant expansion of transduced populations by culture in the presence of hygromycin. Although not increasing transduction efficiency, CD34 enrichment enhanced drug selection, by targeting cells with the greatest self-renewal capacity. Immunoselection of B7.1 expressing cells produced transduced populations with 30–60% expressing B7.1. In an allogeneic mixed leukaemic cell/T lymphocyte reaction (MLLR), transduced AML cells enriched by immunoselection were able to stimulate allogeneic T cells (CD4 and CD8 positive), which could be inhibited by a solubilised B7 receptor, CTLA4.Ig. Our results demonstrate that using a replication incompetent retroviral vector, it is possible to introduce the immune costimulator B7.1 into primary AML blasts and, by immunoselection, enrich the transduced cells, which may be used for subsequent administration as an autologous cellular vaccine.

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.

Conjugation of Lentivirus to Paramagnetic Particles via Nonviral Proteins Allows Efficient Concentration and Infection of Primary Acute Myeloid Leukemia Cells

ABSTRACT Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 109 CFU/ml for vesicular stomatitis virus G protein and 5 × 108 for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and ΔLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.

Influence of interleukin-4 on the phenotype and function of bone marrow-derived murine dendritic cells generated under serum-free conditions

Murine bone marrow‐derived dendritic cells (DC) can be generated by culture in the presence of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) alone or GM‐CSF in conjunction with interleukin‐4 (IL‐4). However, these two culture methods result in the production of heterogeneous DC populations with distinct phenotypic and stimulatory properties. In this study, we investigated the properties of DC generated under serum‐free conditions in the presence or absence of IL‐4 and compared their yield and phenotype to that of DC generated in the presence of fetal calf serum (FCS) (±IL‐4). We did not observe a significant difference in the total cell yield between these four culture conditions, although the proportion of CD11c+ DC in cultures that received FCS was higher than that of their counterparts generated under serum‐free conditions. Also, the four culture conditions generated CD11c+ DC with comparable levels of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86 expression, with the exception of cells cultured under serum‐free conditions in the absence of IL‐4, which displayed suboptimal levels of these markers. Moreover, we compared the functional and stimulatory properties of DC generated under serum‐free conditions in the presence or absence of IL‐4. DC cultured in the presence of IL‐4 were stronger stimulators of allogeneic splenocytes in a primary mixed lymphocyte reaction (MLR) and of naïve antigen‐specific OT‐II transgenic T cells when pulsed with the class II ovalbumin (OVA)323−339 peptide or whole OVA protein than DC cultured in the absence of IL‐4. However, both DC populations displayed a similar capacity to take up fluorescein isothiocyanate (FITC)–albumin by macropinocytosis and FITC–Dextran by the mannose receptor and to secrete IL‐12 in response to stimulation with lipopolysaccharide (LPS) or an agonistic anti‐CD40 monoclonal antibody. Therefore, we conclude that although both DC culture methods result in the production of DC with similar functional abilities, under serum‐free conditions, DC cultured in GM‐CSF and IL‐4 show an increased stimulatory potential over DC cultured in GM‐CSF alone. This is an important consideration in the design of experiments where DC are being exploited as immunotherapeutic vaccines.

In vitro immune modulation by antibodies coupled to tumour cells

Modification of autologous tumour cells to express the immune costimulator B7.1 is a potential strategy for immunotherapy of cancer. Previously, this has involved introduction of genetic material into cells, in vitro culture, and confirmation of the protein product on the cell surface. This is possible only if sufficient tumour is obtainable and efficiently modified in a short time. Whilst progress has been made on ex vivo tumour cell culture and transfection/infection procedures there are still tumour types for which the present means of gene transfer are not efficient enough. We describe a highly efficient in vitro procedure for the modification of over 99% of the cells in a population, allowing the expression of cell surface proteins with potential immune modulatory activities. This procedure, which can be completed in as little as 24 h with no upper limit on cell number, utilizes succinimide esters to label cell surface proteins with biotin covalently. Biotinylated cell membrane proteins then anchor an avidin bridge for immobilizing protein G′-biotin. This can serve to bind immunoglobulin (Ig) molecules via their Fc region such that the variable region of the antibody is freely and functionally available. In the present study the binding of a stimulatory mouse anti-human CD28 monoclonal antibody to the surface of tumour cells is used to show that the modified cells are capable of co-stimulating T cells in vitro. The simplicity of the method, and the use of common reagents, represents a further step towards a realistic, truly ‘off-the-shelf’, nongene immunotherapy protocol.

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

BackgroundAlloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses.ObjectiveTo examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC.MethodsSemi-allogeneic DC were generated from the F1 progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8+ and CD4+ T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo.ResultsIn vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8+ OT-I transgenic T-cells, primed naïve CD4+ OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4+ response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival.ConclusionExpression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Human Liver Regeneration Is Characterized by the Coordinated Expression of Distinct MicroRNA Governing Cell Cycle Fate

In the absence of adequate compensatory regeneration, overwhelming liver damage can cause acute liver failure (ALF) and death without emergent liver transplantation (LT). Auxiliary LT produces satisfactory outcomes in this setting, with the prospect of native liver regeneration sustaining long‐term survival. Since animal models only partially recapitulate human liver regeneration, we investigated the molecular mechanisms controlling it in this unique LT setting, as an exemplar of human liver regeneration. We demonstrate coordinated changes in expression of microRNA (miRNA) during regeneration that drive proliferation, innate immunity and angiogenesis. In contrast, failed regeneration in a similar cohort is associated with distinct miRNA enforcing cell cycle inhibition and DNA methylation. The miRNA expression associated with successful or failed regeneration when recapitulated in vitro, triggered expression of cardinal regeneration‐linked genes promoting cell cycle entry or inhibition, respectively. Furthermore, inhibition of miRNA 150, 663 and 503, whose downregulation is associated with successful regeneration, induced cell proliferation which a key determinant of successful regeneration. Our data indicate that human liver regeneration may be orchestrated by distinct miRNA controlling key regeneration‐linked processes including hepatocyte proliferation. To our knowledge this is the first characterization of molecular processes associated with human liver regeneration.

Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.