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Carolina Herrera, Lilian Rios and Edna Blackman, Advisor: Carol Johnson Messmer High School SMART Team, Milwaukee, WI Faculty mentors: Ashley Conrad, Ph.D. and Bonnie N. Dittel, Ph.D., BloodCenter of Wisconsin, Blood Research Institute, Milwaukee, WI Background Immune System Abstract Multiple Sclerosis (MS) is a disease of the central nervous system (CNS) that commonly affects individuals 20 – 40 years old. MS is thought to be an autoimmune disease in which T cells attack and destroy the myelin sheath surrounding neurons. Demyelinated neurons have a reduced capacity to transmit electrical impulses, causing symptoms from loss of muscle control to memory loss. One protein thought to play a role in MS, is B7-2, a member of a family of proteins that regulate T cell functions expressed by antigen presenting cells (APC). Generation of an immune response by T cells requires two signals: binding of the T-cell receptor to the antigen/MHC complex on APC and binding of B7-1 to CD28 on the T cells. B7-2 is thought to be involved in suppression of the T cell response through binding to CTLA-4. Research using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), demonstrated that injection of B7-2 specific antibodies resulted in a more severe disease course. These data suggest that B7-2 plays a role in negative regulation of the immune response during EAE, possibly by binding CTLA-4. To investigate how B7-2 interacts with CTLA4, we developed a physical model of B7-2 based on its crystal structure (1ncn.pdb) using 3D printing technology that highlights the protein’s β sheet structure and amino acids thought to be important in CTLA-4 binding. This work was supported by a grant from NIH-NCRR-SEPA as part of the SMART team program at Milwaukee School of Engineering. The immune system is composed of many cell types, which are thought to contribute to the onset of MS. The most important being a CD4 T cell. CD4 T cells develop in the thymus and recognize foreign antigens such as bacteria and self-antigens by a process known as antigen presentation. During antigen presentation, antigens are taken up by APC, degraded into small protein fragments called peptides, which are presented on their cell surface bound to MHC molecules. The CD4 T cell binds to the peptide/MHC complex via their cell surface T cell receptor (TCR) (Fig. 2A). For a T cell to become fully activated or responsive (+ signal) to the antigen it must receive a second costimulatory signal delivered by binding of CD28 to one of two B7 (B7-1 or B7-2) molecules expressed by the APC (Fig. 2A). A B APC A CTLA-4 B7 B7 _ + TCR MHC TCR MHC Figure 2. Diagram of antigen presentation and costimulation to T cells. Once activated, T cells enter the CNS and those with the ability to recognize self-antigens expressed by oligodendrocytes become reactivated and initiate an inflammatory response that results in the formation of lesions containing accumulations of numerous immune cells (Fig. 3A) and demyelination (Fig. 3B). B B http://www.health.com/health/library/mdp/0,,zm6056,00.html Summary C Day 0 & 7Immunize with self-antigen Day 0 & 2Pertussis toxin Figure 1. Clinical pathology associated with MS. Diagram of a normal (A,B) and demyelinated neuron (C). MRI image showing a normal (left panel) and MS brain (right panel) (D). Figure 6 B7-2 model showing CTLA-4 binding residues. The binding site between B7-2 and CTLA-4 is thought to be provided by a ring of hydrophobic amino acids (red) on the front face of B7-2 which form a shallow concave surface that allows for the interaction with the MYPPPY loop of CTLA-4. Phe-31 is at the center of the ring, and seems to play a role in the binding properties of B72 to CTLA-4. The aromatic ring of Phe-31 backs up against the pyrrolidine ring of Pro-102 on CTLA-4, giving significant binding energy and presumably stability to the receptor/ligand interaction. The binding interaction is further stabilized through hydrogen bonding of residues Glu-42 and Lys-49 on B7-2 (blue), with the hydroxyl group of Tyr-100 on CTLA-4. Finally, amino acids Leu 38 and Arg -97 (green), provide two beta bulges on the B7-2 molecule that appear to create a better accommodation for the MYPPPY loop of CTLA-4. Figure 3. Pathology in the CNS during a T cell autoimmune attack. Spinal cords from mice with CNS autoimmunity were examined for immune cell infiltration by immunohistochemistry (red staining) (A) and demyelination using immunofluorescence (absence of red staining) (B). In MS, most patients have a relapsing-remitting disease course in which their symptoms spontaneously resolve. The mechanisms leading to this recovery are largely unknown, but have been investigated using the animal model of MS, (EAE). One possible mechanism is the downregulation of the T cell response by the binding of B7 with CTLA-4 on the T cells, delivering a negative signal (Fig. 2B). To examine this possibility, EAE was induced by immunization with self-antigen in adjuvant with pertussis toxin and administered blocking reagents for B7 and CTLA-4 (Fig. 4A). Blocking of CTLA-4 (Fig. 4B) or B7-2 (Fig. 4C) resulted in more severe EAE, suggesting that this interaction is important in controlling autoimmunity in the CNS. A D Figure 5. B7-2 model showing dimerization and β-pleated sheet. The B7-2 model of the human protein is based on its crystal structure (1ncn.pdb.org) (Zhang, et. al., Proc. Natl. Acad. Sci. 100:2586. 2003) and generated using 3D printing technology at the Milwaukee School of Engineering, Center for BioMolecular Modeling, Milwaukee, WI as part of the Students Modeling A Research Project (SMART) program. The SMART team consisted of students from Messmer High School with a school advisor and scientific advisors from BloodCenter of Wisconsin. The model is shown as a dimer as it is thought to bind CTLA-4 and consists of the extracellular domain from residues 1-109 representing the Ig V-type receptor binding domain. The primary structure of B7-2 consists of a twolayer β-sandwich topology shown in orange. T Cell CD28 A C Due to the regulatory role that B7-2 appears to play in regulating CNS autoimmunity, as seen in MS, understanding the structure of B7-2 binding to CTLA-4 on T cells may be critical for the development of therapeutics for MS and other autoimmune disorders. In addition, understanding this ligand/receptor binding may also lead to insights into the structural basis for the signal transduction that occurs intracellulary when B7-2 binds to CTLA-4. We have developed a physical model for the crystal structure of the CTLA-4 receptor binding domain of B7-2 that highlights the amino acid residues in the B7-2 protein shown to play an important role in ligand receptor binding. APC B T Cell Background Multiple Sclerosis MS is believed to be a disease in which the immune system inappropriately attacks oligodendrocytes within the CNS. Oligodendrocytes wrap their cell membranes around neuronal axons generating the myelin sheath (Fig. 1A,B). The immune attack results in damage to the myelin sheath, a process termed demyelination (Fig. 1C). The myelin sheath facilitates nerve transmission along the axon, and when destroyed, alters nerve function. Demyelination is thought to directly contribute to MS clinical symptoms. The autoimmune attack also results in the formation of plaques within the CNS that can be detected by magnetic resonance imaging as white areas (Fig. 1D). B7-2 Model Figure 4. EAE is more severe following the administration of blocking reagents for CTLA-4 or B7-2. EAE was induced by immunization with oligodendrocyte self-antigen in adjuvant on days 0 and 7 accompanied by pertussis toxin injection on days 0 and 2 (A). Blocking of CTLA-4 interactions with B7 by administration of CTLA-4 (B) or anti-B7-2 (C) resulted in more severe EAE as measured on a five point scale as shown on A. Adapted from Racke, et .al. J. Clin. Invest. 96:2195-2203, 1995. MS is a severe, debilitating disease thought to be caused by an autoimmune reaction to the myelin sheath of nerve cells in the CNS. Activation and regulation of an autoimmune response requires presentation of antigen/MHC to T cells as well as binding of B7 proteins, B7-1 and B7-2, to T cells. B7 binding to CD28 activates T cells whereas B7 binding to CTLA-4 inhibits T cell activation. An understanding of how B7-2 binds to CTLA-4 could lead to new medications designed to stop the generation of the autoimmune response in MS patients. A SMART Team project supported by the National Institutes of Health – National Center for Research Resources Science Education Partnership Award