Joanna Galea-Lauri

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.

Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood.

BACKGROUND Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring. METHODS With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population. RESULTS Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation. CONCLUSIONS This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.

Immunological weapons against acute myeloid leukaemia.

A better understanding of the biology of malignant cells and of the host immune system together with dramatic advances in technology have led to the design of innovative immune‐mediated approaches to control neoplastic clones, including various haematological malignancies. One of the major problems with conventional cancer therapies is their inability to eradicate residual cancer cells that are resistant to therapy, hence immune intervention might improve the clinical outcome of patients. This mini‐review will focus mainly on immunological approaches to the therapy of acute myeloid leukaemia (AML), a subset of a much larger family of leukaemias. Immune‐mediated approaches ranging from allogeneic lymphocyte transplants to cytokine therapy, immune‐gene therapy and vaccination by dendritic‐cell‐based vaccines will be discussed.

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.

An immune edited tumour versus a tumour edited immune system: prospects for immune therapy of acute myeloid leukaemia

Cell based therapies for acute myeloid leukaemia (AML) have made significant progress in the last decade benefiting the prognosis and survival of patients with this aggressive form of leukaemia. Due to advances in haematopoietic stem cell transplantation (HSCT) and particularly the advent of reduced intensity conditioning (RIC), the scope of transplantation has now extended to those patients previously ineligible due to age and health restrictions and has been associated with a decrease in transplant related mortality. The apparent graft versus leukaemia (GvL) effect observed following HSCT demonstrates the potential of the immune system to target and eradicate AML cells. Building on previously published pre-clinical studies by ourselves and others, we are now initiating a Phase I clinical study in which lentiviral vectors are used to genetically modify AML cells to express B7.1 (CD80) and IL-2. By combining allogeneic HSCT with immunisation, using the autologous AML cells expressing B7.1 and IL-2, we hope to stimulate immune eradication of residual AML cells in poor prognosis patients that have achieved donor chimerism. In this report we describe the background to cell therapy based approaches for AML, and discuss difficulties associated with the deployment of a chronically stimulated, hence exhausted/depleted immune system to eradicate tumour cells that have already escaped immune surveillance.

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.

A highly sensitive whole blood natural killer cell assay.

The assessment of natural killer cell activity at baseline and the monitoring of this activity during treatment is important in many diseases especially in patients with cancer and AIDS. An optimised and standardised whole blood chromium release assay is described using K562 cells, the standard target erythroleukaemic cell line. The tumour cell lysis observed using whole blood is comparable to that obtained with the standard 4 h lysis assay using peripheral blood mononuclear cells as effector cells. Results with the whole blood assay are reproducible when the incubation with K562 cells is performed over a period of 18 h. The assay necessitates only 0.6 ml of blood collected in 10 IU/ml of sodium heparin as the anticoagulant. In this report, depletion experiments, also standardised using whole blood, show that the effector cells in the whole blood assay are contained within the CD56 + cell population. This assay will be of interest where the immunological status of patients with different diseases need to be frequently monitored.

Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre.

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.