Insights into the manipulation of the immune response to carbohydrate antigens with blood group functional-spacer-lipid constructs
Oliver, Caroline Ann
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The immune system is extremely complex and critical to the survival of all species as it protects them from attack by foreign micro-organisms and viruses. However the immune system can also be a foe in that it can sometimes turn against the organism it is designed to protect resulting in a range of autoimmune diseases. Additionally, its design to protect against invasion of foreign antigens, creates a major hurdle for transfusion and transplantation. If the immune system could be manipulated to allow for transfusion and transplantation of incompatible tissues, or turned off against specific targets in the case of autoimmune disease, all the while maintaining normal protective functions, then major advances in human health and well-being could be made. A variety of approaches have been used to try to manipulate the immune system, but with no one approach being the panacea, thus leaving open the opportunity for novel approaches to try and manipulate it towards creating beneficial outcomes. The recent development of a technology using novel function-spacer-lipid constructs (FSLs) has the ability to modify cell surfaces. As some of these FSLs are related in structure to glycolipids, they have the potential to inhibit antibodies. This research, using mice, set out to determine if FSL constructs could be used to manipulate the immune system by modifying membrane antigens and inhibiting/neutralizing antibodies. By using KODE™ technology, carbohydrate antigens were introduced to red cells (kodecytes) and visualization molecules provided a novel method with which to study and manipulate the immune response. This research involved a series of experiments in mice to investigate antibody stimulation, cell survival measurement and recovery, antibody neutralization and tolerance induction using various FSL constructs. Four hypotheses concerning the use of Functional-Spacer-Lipid (FSL) constructs were developed to investigate their potential for altering the immune response in mice. The hypothesis that FSLs might induce antibody production was tested with FSL constructs to determine the immunogenicity of the molecules with direct infusion into the circulation and subcutaneous immunization. FSL constructs injected by subcutaneous and intravenous routes were not immunogenic, with no anti-A production detected. The hypothesis that kodecytes could be used safely to determine cell survival after transfusion was tested with kodecyte transfusion in both compatible and incompatible mice. Incompatible mice were created by immunization with salivary blood group substance A with the successful production of anti-A and transfusion of A kodecytes. The FSL construct representing blood group A antigen (FSL-A) was intravenously infused into naive and anti-A-positive mice to determine in vivo antigen transformation and cell survival after A antigen-compatible and incompatible (A+biotin kodecyte) transfusions. Other FSL kodecytes were used to create non-lethal, compatible transfusion control mouse models. The FSL-biotin construct provided a label allowing in vitro binding with avidin/Alexafluor 488 providing a fluorescent marker to track kodecyte survival. A method was also developed to recover transfused, circulating A+biotin kodecytes from a whole blood sample with a use of avidin-agarose beads in gel cards. The hypothesis that FSLs could be used to neutralize circulating antibody was tested by infusing FSL constructs to neutralize antibody and then infusing antibody incompatible kodecytes. It was demonstrated that FSL-A was able to neutralize circulating anti-A, mitigating the consequences of kodecyte-incompatible red blood cell transfusion, and allowing for normal cell survival for up to 72 hours. The hypothesis that FSL constructs could potentially induce tolerance was tested by measuring the consequences of infusing FSL-A by direct circulatory infusion into mice, pre-immunization with salivary blood group A substance. Primary immunization with FSL-A did not induce tolerance to the A epitope since anti-A was produced after a secondary immunization of salivary A substance. However, there was some evidence of a partial down-regulation of the immune response in terms of tolerance induction after infusion of FSL-A. With further research, the potential for FSLs to be used safely in humans as a new methodology to determine 24-hour transfused cell survival, or to recover the transfused cells or to neutralize circulating antibody without subsequent antibody stimulation, is now possible.