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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.
A cytotoxic rabbit T-cell line infected with a gamma-herpes virus which expresses CD8 and class II antigens.
A rabbit T-cell line, BJ-610, has been derived from a New Zealand White rabbit infected with Alcelaphine herpes virus-1, which has the characteristics of a lymphokine activated killer (LAK) cell. The surface phenotype of this cell line has been studied by flow cytometry, using a panel of monoclonal antibodies (mAb) to rabbit leucocyte surface markers, and compared with that of another rabbit T-cell line, RL-5, transformed with herpes virus ateles. The expression of a number of markers is common to the two lines; these include the rabbit analogues of CD11a/CD18, CD43, CD44 and CD45. Three antigens are expressed on BJ-610 but not RL-5. One of these is recognized by a mAb thought to recognize CD8, while a second is a class II R-DQ molecule. The third antigen is expressed on thymocytes, a subset of T cells, neutrophils and platelets but its molecular nature is unknown. These two cell lines should prove useful in preparing reagents which recognize subsets of rabbit T cells and for studying the mechanism of herpes virus-induced lymphoid cell deregulation.
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.
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.
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.
Identification and tissue distribution of rabbit leucocyte antigens recognized by monoclonal antibodies.
Three monoclonal antibodies which recognize rabbit leucocytes have been characterized by immunofluorescence staining of a variety of cell populations and also by immunochemical techniques. The evidence obtained suggests that these antibodies recognize the rabbit equivalents of the CD58/LFA-3 (VC21), CD43/leukosialin (L11/135) and CD9 (MM2/57) antigens. A fourth antibody, RPN3/57, recognizes an antigen expressed strongly on T cells, thymocytes and neutrophils and at lower levels on platelets. It has not, however been possible to characterize the antigen recognized by RPN3/57 in molecular terms. Both L11/135 and RPN3/57 are useful reagents for the detection of T cells both by flow cytometry and by immunohistochemistry.
Immunohistochemical identification of leucocyte populations in normal tissue and inflamed synovium of the rabbit.
A panel of monoclonal antibodies which recognizes well-defined markers of rabbit leucocytes has been used in an immunohistochemical survey of normal rabbit spleen, thymus, and liver, and of inflamed synovial tissue. The results demonstrate that these antibodies are well suited for the identification of leucocytes in frozen sections of rabbit tissues and that T lymphocytes, macrophages, and neutrophils can be readily distinguished. Antibodies to CD44 differentiate between medullary and cortical thymocytes, with the cortical cells being almost entirely negative for this marker. Antibodies to RLA class II show that periportal lymphocytes in the liver express RLA-DR but not RLA-DQ. The type A cells of the synovial lining from inflamed joint tissue are stained by antibodies that recognize macrophages in a manner similar to that found in the human synovial lining.
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.
Efficient retroviral infection of mammalian cells is blocked by inhibition of poly(ADP-ribose) polymerase activity.
Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood. However, the endonuclease and DNA strand-joining activities of the virus-encoded integrase protein (IN) are thought to act in concert with other, as-yet-unidentified, endogenous nuclear components which are involved in the DNA repair process. The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which is dependent on DNA strand breaks for its activity, is involved in the efficient repair of DNA strand breaks, and maintenance of genomic integrity, in nucleated eukaryotic cells. In the present work, we examine the possible involvement of PARP in the retroviral life cycle and demonstrate that inhibition of PARP activity, by any one of three independent mechanisms, blocks the infection of mammalian cells by recombinant retroviral vectors. This requirement for PARP activity appears to be restricted to processes involved in the integration of provirus into the host cell DNA. PARP inhibition does not affect viral entry into the host cell, reverse transcription of the viral RNA genome, postintegration synthesis of viral gene products, synthesis of the viral RNA genome, or the generation of infective virions. Therefore, efficient retroviral infection of mammalian cells is blocked by inhibition or PARP activity.
Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo.
OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.
Novel costimulators in the immune gene therapy of cancer.
One of the major goals of cancer immunotherapy is the induction of tumour-specific T-lymphocyte responses that will be effective in the rejection of established tumours. The prospects for such therapy rely on the identification of tumour antigens, and although there is persuasive evidence for the presence of such antigens,1,2 the occurrence of the disease does illustrate that the immune system is at least, on some occasions, unable to recognise and destroy these targets. Tumour antigens may be novel proteins (from genetic lesions or viral infections), modified existing antigens (eg, abnormally glycosylated cell surface proteins), or inappropriately expressed normal gene products (eg, CA125, carcinoembryonic antigen, and alpha-fetoprotein).1 Involvement of the immune system in the normal surveillance and suppression of cancer is further suggested by the increased incidence of tumours in immunocompromised patients.3 However, recent evidence has shown that, at least in model systems, cancer cells can be modulated in such a way that they stimulate cells of the immune system to recognise and destroy these malignant cells. This review summarizes the costimulatory molecules involved in the activation of such cells, the principles and mechanisms underlying their activation, and how such knowledge can be used to persuade the immune system to challenge cancer.
Streptavidin paramagnetic particles provide a choice of three affinity-based capture and magnetic concentration strategies for retroviral vectors.
Three strategies have been designed to concentrate infectious retroviral vectors from the supernatants of human- (HT1080) and murine- (NIH 3T3) based packaging cells. Streptavidin-conjugated paramagnetic particles in conjunction with (i) antibodies directed against murine fibronectin, (ii) biotinylated lectins, or (iii) biotin-modified packaging cell-surface proteins allow affinity-mediated magnetic concentration of retroviral vectors. Retroviral titers (assayed by colony formation of human myeloid K562 cells) are increased by 1-4 x 10(3)-fold after volume reductions of only 125-fold. Using these procedures, preparations of 5 x 10(8) cfu/ml are routinely made from relatively low-titer (2-5 x 10(5) cfu/ml) starting material. High-titer (paramagnetic) retroviral vector preparations can be used for magnetic field-dependent retroviral infection in vitro. Magnetic field-dependent localization such as this may enable the in vivo administration of formulations that concentrate retroviral infection to the required target tissues and organs.
The expression of CD11/CD18 molecules on rabbit leucocytes: identification of monoclonal antibodies to CD18 and their effect on cellular adhesion processes.
The monoclonal antibodies, L13/64 and RCN1/21, raised against rabbit leucocytes, have been shown, by sequential immunoprecipitation and immunoblotting, to react with the rabbit CD18 molecule. They recognise not only surface-expressed CD18 but also an intracellular form which appears to be partially glycosylated. The expression of the CD11 and CD18 glycoproteins on a wide variety of rabbit leucocyte populations has been investigated by flow cytometry, using these two monoclonal antibodies (Mabs), together with others which recognise human CD11 and CD18 proteins but cross-react with rabbit tissues. The distribution of these leucocyte integrin molecules has been shown to be similar to that observed in humans and determination of the N-terminal sequence of rabbit CD11b shows strong homology with human and mouse sequences. Of four anti-rabbit CD18 Mabs tested, only one, L13/64, has been shown to be capable of inhibiting the adhesion of fMLP-stimulated neutrophils to gelatin coated plastic and the homotypic aggregation of PMA-stimulated T cells, both of which assays have been shown to be CD18-dependent. RCN1/21 causes aggregation of unstimulated neutrophils, but it is not known whether this is due to cellular activation or agglutination.
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 naive 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.
IL-2/B7.1 (CD80) fusagene transduction of AML blasts by a self-inactivating lentiviral vector stimulates T cell responses in vitro: a strategy to generate whole cell vaccines for AML.
Combined expression of costimulatory factors and proinflammatory cytokines stimulate effective immune-mediated tumor rejection in a variety of murine tumor models. Specifically, syngeneic tumor cells genetically modified to express B7.1 (CD80) have been shown to induce rejection of previously established murine solid tumors, and transduction with IL-2 can further increase survival. However, poor rates of gene transfer and inefficient expression of multiple transgenes encoded by single vectors have hampered the development of such autologous tumor cell vaccines for clinical trials in acute myeloid leukemia (AML) patients. Here we describe the development of a self-inactivating lentiviral vector encoding B7.1 and IL-2 as a single fusion protein postsynthetically cleaved to generate biologically active membrane-anchored B7.1 and secreted IL-2. This enables the efficient transduction of both established and primary AML blasts, resulting in expression of the transgenes in up to 98% of the cells following a single round of infection at an m.o.i. of 10. The combined expression of IL-2 and B7.1 in AML blasts enables increased stimulation of both allogeneic and autologous T cells. The stimulated lymphocytes secrete greater levels of Th1 cytokines and show evidence of specificity, as indicated by their increased proliferation in the presence of autologous AML compared to remission bone marrow cells.
Suppression of intra-articular responses to interleukin-1 by transfer of the interleukin-1 receptor antagonist gene to synovium.
We have developed an ex vivo method for delivering genes to the synovial lining of joints and expressing them intra-articularly. The present studies were designed to determine whether transfer of a human interleukin-1 receptor antagonist protein (IRAP) gene by this method was able to antagonize the intra-articular actions of interleukin-1. Intra-articular injections of human recombinant interleukin-1 beta (hrIL-1 beta) into the knees of control rabbits provoked a marked leukocytic infiltrate into the joint space, severe synovial thickening and hypercellularity, and loss of proteoglycans from articular cartilage. Genetically modified knees contained several nanograms of human IRAP and inhibited each of these effects of IL-1 beta. These data demonstrate for the first time that delivery of an appropriate gene to joints can prevent intra-articular pathology. Such findings permit cautious optimism about the eventual development of a gene treatment for arthritis and other disorders of the joint.