Immune Stimulatory Capacity Research Articles

Tumour-dendritic hybrid cell vaccination for the treatment of patients with malignant melanoma: immunological effects and clinical results

Hybrid cell vaccines of autologous tumour cells fused with allogenic dendritic cells (DC) combine the tumour's antigenicity with the immune-stimulatory capacity of mature dendritic cells and allogenic MHC class II molecules to activate T cell help and induce tumour-specific cytotoxic T cells. This concept was tested in a clinical trial with melanoma stage III and IV patients. Seventeen patients were evaluated: one experienced complete, one partial response and six stable disease with long survival times. Eleven of fourteen patients, clinical responders and non-responders alike, mounted high-frequency T cell responses to various tumour-associated antigens. Failing clinical responses correlated with loss of antigenicity.

Stimulation of Precursor-B Acute Lymphoblastic Leukemia Cells with Toll-Like Receptor Ligands Alters Their Immunogenicity.

Pediatric pre-B acute lymphoblastic leukemia is the most common childhood cancer. Although current therapies achieve a high rate of remission, relapse of pre-B ALL remains a significant clinical challenge and new forms of therapy are needed. The graft-vs-leukemia (GVL) effect after bone marrow transplantation has shown that the immune system is capable of producing an effective anti-tumor response, which suggests that immune-mediated therapies may provide a complementary treatment strategy. Toll-like receptors (TLR), found on many immune cells, including B cells, have been shown to be important molecules in both innate and adaptive immune responses. Ligation of TLRs with their respective ligands results in an increase in the immunogenicity of antigen presenting cells (APC), through upregulation of MHC antigens and costimulatory molecules and production of cytokines and chemokines. We have previously reported that stimulation of pre-B ALL cells with the TLR9 ligand, CpG DNA, enhances the induction of Th1 immune responses by allogeneic T cells. In this study we examined the expression profile of TLRs 1-8 in precursor-B ALL cells and the effects of TLR ligation on the immune stimulatory capacity of precursor-B ALL cell lines. Eight precursor-B ALL cell lines were used in this study (Nalm6, REH, Sup-B15, KOPN-57bi, 380, 697, OP-1 and RS4:11). Standard RT-PCR analysis was used to examine the expression of TLRs 1-8 by the cell lines. The cell lines were stimulated with ligands for TLR2 (peptidoglycan), TLR3 (poly I:C) and TLR4 (LPS) and evaluated for changes in costimulatory molecule expression and allogeneic T cell stimulation. Non-quantatative PCR detected each TLR, with the exception of TLR8, in the majority of the cell lines. TLR8 was only detected, at low level, in RS4:11 cells. Despite the broad expression profile of the TLRs, significant differences in the effect of TLR ligation were observed between cell lines. In general, only modest increases in CD40 and CD86 expression were observed on responsive cell lines, with the majority of the lines showing no significant changes in response to TLR2, 3 or 4 ligation, despite receptor detection by PCR. Changes in allogeneic T cell proliferation in response to TLR stimulated ALL cell lines were observed, with the largest increases occurring with peptidoglycan and LPS. As was the case with costimulatory molecule expression, no T cell proliferative response change was common to all cell lines. However, analysis of cytokine production by T cells revealed a consistent increase in IFN-gamma and reduction in IL-5 levels in response to peptidoglycan stimulated ALL cells. The results reported in this study indicate that precursor-B ALL cell lines express several TLR molecules and that TLR ligation alters the immunogenicity of the majority of these lines. Interestingly, ligation of TLR2 with peptidoglycan induced a profound shift towards Th1 cytokine production. These observatios suggest that TLR ligation, most notably TLR2 ligation, may provide a strategy to influence anti-ALL immune responses and enhance immune mediated control of this disease.

Effect of incorporation of the adjuvant Quil A on structure and immune stimulatory capacity of liposomes.

Liposomes have been widely used as drug delivery systems for many years. However, they are of limited use as delivery systems for subunit vaccines due to their low immunogenicity. Here we examine the effect of incorporating the adjuvant Quil A into liposomes on the type of particles produced, on the ability of the different particles to incorporate antigen and on the ability of the different particles to stimulate murine bone-marrow-derived dendritic cells (DC) and lymphocytes. The incorporation of increasing amounts of Quil A, from 20% to 70% of the total lipid into liposomes, reduces the size of the particles that form in aqueous dispersion and decreases antigen incorporation and uptake by DC. Interestingly, the particles with 20% Quil A were more toxic to cells in culture than the particles containing 70% Quil A, and the 20% particles were also more immunostimulatory.

The use of lentiviral vectors in gene therapy of leukemia: combinatorial gene delivery of immunomodulators into leukemia cells by state-of-the-art vectors

Our goal is to develop cell vaccines against leukemia cells, genetically modified to express molecules with potent immune-stimulatory capacities. Pre-clinical evaluation of this approach in murine models has demonstrated efficient anti-leukemic responses with the expression of immunomodulators, in particular GM-CSF and CD80, in irradiated cell vaccines. We have previously shown efficient insertion of GM-CSF and CD80 genes into primary human leukemia cells with the use of second and third generation self-inactivating (SIN) lentiviral vectors (Blood 96 (2000), 1317; Leukemia 16 (2002), 1645). The advantages of lentiviral vectors for development of autologous leukemia cell vaccines include: (1) efficient and consistent gene delivery; (2) high levels of transgene expression; (3) persistent expression of the transduced gene; (4) no viral proteins, as only the transduced gene is expressed; (5) no undesirable cytotoxic effects, and; (6) simplicity of use [leukemia cells are exposed to vector(s) only once]. In this work, we evaluated the insertion of the central polypurine tract and the central termination sequence into a SIN lentiviral vector encoding for GM-CSF and CD80, which significantly enhanced the transduction efficiency of primary leukemia cells and provided higher levels of GM-CSF and CD80 co-expression. We also demonstrate a methodology to deliver simultaneously a combination of immunomodulatory molecules (GM-CSF, CD80, IL-4, and CD40L) to activate different pathways of immune stimulation. Therefore, lentiviral vectors offer a simple, versatile, and reliable approach for engineering leukemic cells for use as cell vaccines.

Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells.

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.

In vivo and in vitro Modulation of Immune Stimulatory Capacity of Primary Dendritic Cells by Adenovirus-Mediated Gene Transduction

Dendritic cells (DCs) are potent antigen-presenting cells which are key leukocytes in the initiation of cell-mediated organ graft rejection, antiviral immunity, and antitumor responses. In this study we demonstrate that genetic modification of primary human and mouse DCs by adenoviral gene transfer is an effective means of induction and modulation of antigen presentation by DCs. An adenovirus vector (AdLacZ) was used to express an intracellular model antigen β-galactosidase (β-gal) in DCs. Our results show that 30–40% of precursor dendritic cells (PDCs) derived from human umbilical cord blood and circulating mature blood DCs express high levels β-galactosidase (β-gal) after infection with AdLacZ with no cytopathic effect observed. In vitro, AdLacZ transduced PDCs and DCs demonstrated a 10- to 20-fold higher mixed lymphocyte reaction (MLR) stimulatory capacity as compared to that of monocytes. In vivo, immunization with AdLacZ transduced mouse DCs resulted in more potent cytotoxic T lymphocyte (CTL) responses against the predicted H-2 restricted β-gal epitope as compared to CTL responses obtained by β-gal peptide pulsed DCs. Modulations of the MLR stimulatory capacity of DCs were examined by expression of mouse B7 and CTLA-4Ig. The results show that expression of mouse B7 by a recombinant adenoviral vector (Ad7) significantly enhances the MLR stimulatory capacity of human DCs. In contrast, expression of CTLA-4Ig (AdCTLA-4Ig) reduces the MLR stimulatory capacity of the transduced cells. We conclude that recombinant adenovirus can readily be used for genetic modulation DC-induced immune responses in vivo and in vitro. DCs targeted for induction of specific antigen responses or for modulation of the immune stimulatory capacity may have a potential use in the control of transplantation rejection or viral infections.

Lysophosphatidylcholine is involved in the antigenicity of oxidized LDL.

Lysophosphatidylcholine (LPC) is formed by hydrolysis of PC in low density lipoprotein (LDL) and cell membranes by phospholipase A2 or by oxidation. Oxidized (ox) LDL activates endothelial cells, an effect mimicked by LPC. oxLDL also has the capacity to activate T and B cells, and antibody titers to oxLDL are related to the degree of atherosclerosis. The antigen in oxLDL responsible for its immune-stimulatory capacity is not well characterized, and we hypothesized that LPC was involved. We demonstrate herein the presence of antibodies against LPC, both of the IgG and IgM isotype, in 210 healthy individuals. This antibody reactivity was not specifically related to oxidation of the fatty acid moiety in LPC, since LPC containing only palmitic acid showed antibody titers equivalent to those of LPC containing unsaturated fatty acids. Antibody titers to PC were low compared with LPC, and hydrolysis of PC at the sn-2 position is thus essential for immune reactivity. There was a close correlation between anti-oxLDL and anti-LPC antibodies. Furthermore, LPC competitively inhibited anti-oxLDL reactivity, which indicates that LPC may explain a significant part of the immune-stimulatory properties of oxLDL. LPC, being a lipid, is not likely to be an antigen itself. Instead, LPC could form immunogenic complexes with peptides, which may induce and potentiate immune reactions in the vessel wall. This study adds to the evidence that LPC is an important component of oxLDL and emphasizes the potential role of phospholipase A2 in atherosclerosis.