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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