Increased immunogenicity of surviving tumor cells enables cooperation between liposomal doxorubicin and IL-18

<p>Abstract</p> <p>Background</p> <p>Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated.</p> <p>Methods</p> <p>Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack <it>in vitro</it>. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated <it>in vivo </it>in mice bearing ID8-Vegf tumors.</p> <p>Results</p> <p>While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death <it>in vitro</it>. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy <it>in vivo </it>and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice.</p> <p>Conclusion</p> <p>These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 <it>in vivo</it>.</p

Distinct Effects of IL-18 on the Engraftment and Function of Human Effector CD8+ T Cells and Regulatory T Cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8+ effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Rα was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies

Expression and functional analysis of murine intercellular adhesion molecule 1 (ICAM-1)

Cell adhesion molecules enhance Interactions between adjacent cells In order to mediate a large variety of functions of the Immune system. An antibody against the murine lymphocyte surface antigen MALA-2 has previously been shown to Inhibit mixed lymphocyte response. A λgt10 cDNA library from NS-1 cells was screened and a cDNA clone, K3-1.1, was previously isolated. It had significant homology to the human ICAM-1 gene. This thesis covers the isolation of a second cDNA clone, K4-1.1, and its comparison to K3-1.1 In terms of expression, function and distribution. The two clones are identical in sequence with the exception of the 5’ ends. Expression of these two clones was examined using a transient expression system of COS cell transfection. Cell surface expression of the K3-1.1 clone could not be detected by FACS analysis. Even when the 5' untranslated region of the K3-1.1 clone (which has 10 potential translation start sites) was removed, protein could not be detected at the cell surface, intracellularly, or extracellularly. However, K4-1.1 expression was detected at the cell surface. Northern blot analysis reveals that there are two distinct messages which are likely to be represented by the two clones. When the northern blot was probed with the 5' end of the K3-1.1 clone, only one of the messages was detected. This together with the result of Southern blot analysis suggests that the two messages are likely the result of alternate splicing. In order to examine the interactions of the murine ICAM-1 with the surface of other cells, an expression system which would produce large amounts of a secreted soluble form was established. The soluble protein was purified from the supernatant of transfected cells by an antibody-affinity column and used in preliminary binding assays.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Role of intercellular adhesion molecule 2(ICAM-2) in the murine immune system

Intercellular adhesion molecule-2 (ICAM-2; CD102) is one of three ligands for the p2 leukocyte integrin LFA-1 (CD11a/CD18). Although ICAM-2 expression is limited to lymphocytes, monocytes, granulocytes, and endothelium, the biological role of ICAM-2 has remained unknown. In this thesis, the murine ICAM-2 cDNA was cloned in order to assist the functional investigation. Sequence analysis of both the cDNA and the genomic clone revealed that ICAM-2 is a member of the immunoglobulin superfamily. The cDNA and antibody were used to examine the role of ICAM-2 in T cell activation and leukocyte transendothelial migration. In order to examine the role of ICAM-2 in antigen presentation to T cells, murine fibroblastic L cells expressing allogeneic class II MHC (l-Ed) were transfected with the ICAM-2 cDNA and tested for the ability to stimulate splenic T cells. The expression of ICAM-2 significantly increased the stimulation of T cells in an LFA-1-dependent manner. The increased T cell response was also observed when the class II MHC and ICAM-2 were expressed in separate cells combined together. This indicated that ICAM-2 is actually transmitting a costimulatory signal rather than merely enhancing T cell adhesion to the antigen presenting cell. T cells stimulated with ICAM-2-transfected L cells expressing the class II MHC were able to respond to an allogeneic secondary stimulation. In contrast, T cells stimulated with L cells expressing only the class II MHC were not able to respond to allogeneic stimulation in the secondary response. These results indicated that ICAM-2 may provide a necessary costimulatory signal to the T cell that is required for the aversion of an anergic state. Endothelial cells transfected with the ICAM-2 cDNA were examined for the ability to assist leukocyte migration. A system was set up in which transendothelial migration could be easily quantitated. It was found that a lymphocytic cell line and bone marrow neutrophils were able to utilize ICAM-2 for the migratory process without destroying the endothelial monolayer. These results demonstrate that ICAM-2 is able to play a role in two physiological processes that are of central importance to the normal function of the immune system.Medicine, Faculty ofMedical Genetics, Department ofGraduat

YQB. Québec à la conquête de l’air, Quebec City. The Sky’s the Limit!

Previous studies have demonstrated that CD44 isoforms containing the alternatively spliced exon v10 promote cell-cell adhesion via a mechanism that involves the recognition of chondroitin sulfate side chains presented on the surface of interacting cells in association with other CD44 molecules. Sequence analysis revealed the presence within exon v10 of two motifs that may be relevant to this interaction, a B[X(7)]B motif that may contribute to the recognition and binding of chondroitin sulfate and a serine-glycine motif that may serve as a site of chondroitin sulfate attachment. To determine whether either of these two motifs explain the unique adhesive activity of exon v10-containing CD44 isoforms, each was targeted by site-directed mutagenesis, and the adhesive activity of the resultant mutants was determined using a quantitative cell-cell binding assay. The data obtained demonstrate conclusively that it is the exon v10-encoded B[X(7)]B motif that is solely responsible for the enhanced adhesive activity of exon v10-containing CD44 isoforms

Identification and Characterization of CD44RC, a Novel Alternatively Spliced Soluble CD44 Isoform that can Potentiate the Hyaluronan Binding Activity of Cell Surface CD44

AbstractSoluble CD44 proteins generated by proteolytic cleavage or aberrant intron retention have been shown to antagonize the ligand binding activity of the corresponding cell surface receptor, inducing apoptosis and inhibiting tumor growth. Interestingly, such findings appear to contradict recent studies demonstrating a correlation between the presence of high levels of soluble CD44 in the serum of cancer patients and poor prognosis. In the present study, we report the cloning of a novel, naturally occurring, differentially expressed, soluble CD44 isoform, designated CD44RC, which, in contrast to previously described soluble CD44 proteins, can dramatically enhance the hyaluronan binding activity of cell surface CD44. Sequence analysis suggests that CD44RC is generated by an alternative splicing event in which the 3′ end of CD44 exon 2 is spliced into an internal splice acceptor site present within exon 18, altering reading frame and giving rise to a soluble protein with a unique COON terminus. Functional studies suggest that CD44RC enhances hyaluronan binding by adhering to chondroitin sulfate side-chains attached to cell surface CD44, generating a multivalent complex with increased avidity for hyaluronan