Molecular Mechanisms of Immunosuppression
Studies of Molecular Mechanisms of Ganglioside-Induced Immunosuppression
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DID/Diagnostics & Devices/Medicine
8 Project completed
Senior Project Manager
Weaver L M
Institute of Bioorganic Chemistry, Russia, Moscow
- Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- University of Milan / Department of Medical Chemistry and Biochemistry, Italy, Milan\nUS Department of Health & Human Services / National Institute of Health / National Cancer Institute / Division of Clinical Sciences, USA, MD, Bethesda\nInstitute of Physical and Chemical Research (RIKEN), Japan, Saitama, Wako\nUniversity of Minnesota / The Hormel Institute, USA, MN, Austin
Project summaryGangliosides are a unique class of complex acidic glycolipids located ubiquitously in the outer leaflet of the plasma membrane of mammalian cells. They have a multitude of functions in maintaining immune homeostasis and regulating immune responses, including cell recognition, cell adhesion, and cell growth control. Gangliosides are capable of both enhancing and attenuating the antigen-generated immune response. Ganglioside shedding by tumor cells (neuroblastoma, melanoma, lymphoma, mammary carcinoma, hepatoma etc.) is a well-known process, and a number of gangliosides have been defined as clinical tumor markers. One result of this shedding is a high level of tumor-specific gangliosides in serum (e.g. gangliosides GM2 and GD2). Gangliosides shed by rapidly growing tumors of the above-mentioned types may be the major contributors to the generalised immunosuppression often observed in cancer patients. One of the major mechanisms of this type of immunosuppression may be the direct ganglioside interaction with the cytokines that play the crucial role in the T cell response. The primary mechanism of ganglioside suppression of IL-2- and IL-4-dependent processes is the competition between gangliosides and IL-2- and IL-4-receptors for interleukin binding.
One of the existing approaches to immune system enhancement in cancer patients is treatment with recombinant IL-2 and IL-4 (rIL-2 and rIL-4). Nevertheless, the presence of high levels of shed gangliosides in the bloodstream can reduce the amount of free IL-2 and IL-4 available for receptor-mediated signaling. This unfortunate characteristic of ganglioside shedding makes higher doses of cytokine necessary to overcome this blockade, increasing the risk of toxic side effects associated with rIL-2 and rIL-4 treatment. Although several schemes have been proposed to both limit ganglioside binding and cytokine toxicity (including encapsulation of rIL-2 in liposomes), these alternatives are not particularly effective in either reducing ganglioside blockade of IL-2/IL-4 function or lowering the cytokine dosage required to enhance cell-mediated immunity.
We suggest an alternative, direct approach for the immunity boosting in cancer patients: to bind free gangliosides by synthetic peptides corresponding to the ganglioside binding site(s) on IL-2 and IL-4. Pretreatment with such peptides would presumably reduce ganglioside sequestration of cytokine and increase systemic IL-2 and IL-4 levels. To develop this method, it is necessary to know the structure of ganglioside-binding sites of IL-2 and IL-4 molecules. Over the past two decades, there have been numerous reports indicating the existence of such ganglioside-binding abilities and structures in the IL-2 and IL-4 molecules but the primary sequences of such sites have never been determined. We therefore propose to precisely define the mechanism of ganglioside-cytokine binding, identify the relevant cytokine binding domains using newly synthesized ganglioside photoaffine derivatives, generate peptides corresponding to those domains, and evaluating their ability to block ganglioside-cytokine interactions. We then propose to measure the therapeutic efficacy of these peptides in several mouse models of tumor development and autoimmunity.
This project will consist of four objectives:
1. Ganglioside influence on T lymphocyte functions. Using a mathematical model, we have previously defined that gangliosides GM3, GT1b and GD3 inhibit IL-2- dependent proliferation of CTLL-2 cells (or IL-4-dependent proliferation of CT.4R cells) as competitive inhibitors while GM1 is a mixed-type inhibitor. In this study, the inhibition activities of ganglioside tumor markers GM2 and GD2 will be similarly accessed for type of inhibition and dissociation constants using 1. fluorescent-labeled gangliosides incorporated into liposomes.
2. Ganglioside/interleukin binding sites. The ability of gangliosides GM1 and GM3 to bind IL-2 and IL-4 and to inhibit IL-2- and IL-4-dependent cell proliferation has been previously established. The precise site of ganglioside binding to IL-2 and/or IL-2-receptor is presently unknown. To determine the interaction site, we will use ganglioside derivatives labeled with photoaffine diazocyclopentadiene-2-carbonyl group and iodine-125. The label will be either in the acyl residue to study IL-2(4) receptor/ganglioside interaction, or in the polar head to analyze lymphokine/ganglioside interaction. The primary structures of ganglioside binding site(s) in IL-2 and IL-4 molecules will be determined by protein sequencing and mass spectrometry.
3. Effects of the ganglioside binding site peptides on cytokine-dependent T cell line function. Peptide(s) corresponding to ganglioside binding site(s) in IL-2 and IL-4 molecules will be synthesized and their ability to block ganglioside sequestration of cytokines will be measured using IL-2- and IL-4-dependent T cell lines. Level of cell proliferation in the presence of ganglioside, cytokine and blocking peptide will be measured using these indicator cell systems.
4. Effects of ganglioside binding site peptides on peripheral T cell proliferation and differentiation and augmentation of IL-2 immuunotherapy. If the peptide products are shown to relieve suppression of IL-2 or IL-4 binding to their cognate receptors, they will be evaluated in several in vitro models of normal T cell activation by mitogens and suppression by tumor cell supernatants. They will also be evaluated for efficacy in several established in vivo systems, including a transplanted mammary tumor mouse model, a transplanted B6 melanoma mouse model.