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Cloning an Army of T Cells for Immune Defense
Introduction
The cells of our immune system discover and destroy foreign invaders
that enter our bodies and may threaten our health. Among the invaders
are viruses, bacteria, and other microscopic pathogens. Pathogens are
recognized by the immune system as not being part of the body, as "non-self."
The immune system probes specific proteins on the surface of invading
microbes, first recognizing the proteins as foreign and then coordinating
their destruction by a variety of strategies, including producing antibodies
and engulfing foreign cells.
Immune-system cells defend our bodies by acting as a coordinated team.
This cellular defense team communicates in precise, highly regulated ways.
Specific molecules on their surfaces mediate the communication among immune
system cells. Although highly specific, the interactions and responses
of the immune system depend on chance meetings of cells in the fluid spaces
of the lymphatic system and circulatory system.
View the animation to see how one type of immune cell—the helper
T cell—interprets a message presented at the surface of the cell
membrane. The message is an antigen, a protein fragment taken from an
invading microbe. A series of events unfolds that results in the production
of many clones of the helper T cell. These identical T cells can serve
as a brigade forming an essential communication network to activate B
cells, which make antibodies that will specifically attack the activating
antigen.
The animation illustrates several fundamental biological principles and
processes common to many cellular functions. It may be helpful to view
the animation several times, first to gain a general impression of the
signal transduction process and then to focus on particular molecular
details.
Four main themes emerge:
- Signal transduction. Molecular information is presented at
the surface of the T cell. The molecular message is then processed within
the T cell through a complex cascade of molecular interactions called
signal transduction. The molecular cascade stimulates a reaction in
the receiving T cell: the production of specific receptors and extracellular
signaling molecules.
- Signal amplification. When a cell processes a message, it often
also amplifies that message. At each step in the molecular cascade,
the signal may become stronger. In the case of the T cell, it will release
many molecules of the protein interleukin-2 (IL-2) and produce many
IL-2 receptors, and in the end, many thousands of identical T cells
will be produced.
- Molecular switching. Slight changes to the state or shape of
a protein can convey information or trigger a cellular process. Molecules
can be turned on and off through molecular switches. Adding and removing
a phosphate group—called phosphorylation and dephosphorylation,
respectively—are important and common examples of changes that
can flip a molecular switch.
- Molecular specificity. Molecules interact with one another
in specific ways. Enzymes will only work on certain substrate molecules.
Receptors will only bind certain ligands. Only specific sets of molecules
will associate to form molecular complexes or gather at particular docking
sites to do their job.
 Part
1
Triggering the Cascade: Foreign Antigen Activates T-Cell Receptor
Millions of different T cells are present in the lymphatic tissue. Each
is poised to rapidly reproduce if triggered by the right molecular signal.
The signal is presented by a special cell called the antigen-presenting
cell (APC). The APC carries a foreign antigen, a piece of protein from
an invader. The antigen is attached to a special molecule called MHCII
(major histocompatibility complex II). The T cell and the APC bind to
each other via the MHCII-antigen complex and a receptor on the T cell
called the T-cell receptor (TCR). The binding initiates the passage of
signals across the T-cell membrane. The TCR molecule spans the T-cell
membrane, and some of its parts, or subunits, are within the cell and
others project outside the cell. Within the T cell is a subunit of the
TCR that contains many tyrosine amino acids (circles). This subunit is
called the ITAM (immunoreceptor tyrosine-based activation motif). The
ITAM is an important activation site for molecules that are essential
for signal transduction to proceed.
Summary of the action in Part 1
- The T cell and APC come into contact.
- The MHCII-antigen complex and TCR bind.
- Binding initiates a series of events focused around the ITAM portion
of the TCR.
 Part
2
Activating Important Molecules Near the Membrane: ITAM, ZAP-70, and
Other Kinases
To be activated and work together, the molecules contributing to the
transduction cascade need to be present together in the correct part of
the cell. The inactivated molecule ZAP-70 (zeta-associated protein-70),
a key player in the cascade, may randomly bump into the ITAM subunit of
the T-cell receptor. However, nothing will come of these contacts unless
ITAM is in the activated state. The binding of the APC to the T-cell receptor
triggers a series of events leading to ITAM activation.
- The plasma membrane of the T cell reorganizes in such a way that the
coreceptor CD4 and an enzyme called Lck move close to ITAM. Lck is a
kinase, an enzyme that activates other molecules by adding a phosphate
group (a process called phosphorylation).
- Lck phosphorylates tyrosine residues in ITAM, activating the ITAM
by changing its conformation.
- With ITAM activated, ZAP-70 will now bind upon contact. Specifically,
a special region of ZAP-70 called the SH2 (Src homology 2) domain (yellow
region) binds to the phosphorylated ITAM docking site.
- Now ZAP-70 is in a position to also be phosphorylated by Lck.
- ZAP-70, another kinase, is activated by phosphorylation.
Summary of the action in Part 2
- ZAP-70 contacts ITAM but fails to bind because ITAM is not activated
(phosphorylated
- The plasma membrane reorganizes, bringing Lck, associated with CD4,
close to ITAM. Lck phosphorylates ITAM.
- Phosphorylated ITAM region serves as a docking site for ZAP-70.
- ZAP-70 binds to phosphorylated ITAM.
- Lck phosphorylates ZAP-70.
- ZAP-70 is activated.
Part 3
The Message Moves into the Cell: Membrane Complexes Transmit the Signal
into the Cytoplasm
The cascade of events at the T-cell membrane transmits the signal farther
into the cell. At this stage, a molecule called LAT (linker for activation
of T cells) will be modified to act as a docking site to bring other important
proteins close to the cell membrane.
- A molecule in the cytoplasm called phospholipase C-gamma (PLCg)
has the ability to act on phospholipids in the membrane to convert phosphatidylinositol
bisphosphate (PIP2) into inositol triphosphate
(IP3). IP3 is an important
signaling molecule within cells.
- PLCg is brought into contact with the membrane
phospholipids by docking to the long membrane-spanning molecule LAT.
However, PLCg will not dock to inactivated
LAT.
- Once Lck phosphorylates ZAP-70, ZAP-70 can phosphorylate LAT.
- Now PLCg, through its SH2 domain (yellow
region), can dock onto LAT.
- Subsequently, PLCg is phosphorylated (white
circle) and activated (red region), leading to the release of IP3
into the cell's cytoplasm.
Summary of the action in Part 3
- PLCg fails to dock to LAT because no phosphorylated
tyrosine residues are present.
- Activated ZAP-70 phosphorylates tyrosine (circle) in LAT that is closest
to membrane.
- PLCg docks onto phosphorylated tyrosine
in LAT.
- PLCg is phosphorylated and activated.
- PIP2 within the T-cell membrane is catalyzed
by activated PLCg into IP3
molecules.
- IP3 moves away from the membrane and into
the cytosol.
 Part
4
Making Waves: Cytoplasmic Signals Cause the Release of Calcium
IP3 acts as a messenger to deliver the signal
received by the T-cell receptor from the cell membrane to the endoplasmic
reticulum (ER). The ER, a cellular organelle with its own membrane, serves
as a large reservoir of calcium ions (Ca++).
Ca++ is another important cellular signaling
molecule. IP3 spreads throughout the cytoplasm
but binds specifically to specialized receptors on the surface of the
ER. IP3 binding to its ER receptor causes channels
in the ER membrane to open and stored Ca++
is released in waves that spread throughout the cell. Ca++
forms a complex with calmodulin and calcineurin, two proteins named for
their ability to partner with calcium. Once the complex is formed, Ca++/calmodulin/calcineurin
can in turn act to dephosphorylate the transcription factor NF-AT (nuclear
factor of activated T cells). Transcription factors are molecules that
can directly affect the expression of genes, turning them up or down.
NF-AT must enter the nucleus in order to affect gene expression, but in
it's phosphorylated state (white circle) it cannot. However, once dephosphorylated
(gray circle) by the Ca++/calmodulin/calcineurin
complex, NF-AT can pass through the nuclear pores and into the T cell's
nucleus.
Summary of the action in Part 4:
- IP3 binds to specific receptors on the ER.
- Ca++ is released from the ER in waves that
travel throughout the cytoplasm.
- Ca++ forms a complex with calmodulin and
calcineurin.
- The transcription factor NF-AT is unable to enter the nucleus when
in a phosphorylated form (white circle).
- The Ca++/calmodulin/calcineurin complex
dephosphorylates NF-AT.
- NF-AT enters the nucleus.
Part 5
Turning on Genes: Initiation of IL-2 Gene Transcription
Within the nucleus, NF-AT, along with other transcription factors (diamond
and rectangle) not detailed in the animation, binds to the promoter region
of the interleukin-2 (IL-2) gene to initiate gene transcription. IL-2
messenger RNA (mRNA) is produced in the nucleus and exported to the cytoplasm,
where IL-2 protein is translated in the ribosome. IL-2 protein is further
processed in the ER and Golgi and then exported out of the cell.
Summary of the action in Part 5:
- NF-AT and other transcription factors activate transcription of the
IL-2 gene.
- IL-2 mRNA is produced and exported out of the nucleus.
- Ribosomes translate the IL-2 mRNA into protein.
- IL-2 protein is processed in the ER and Golgi.
- IL-2 protein is exported out of the T cell.
Part 6
An Army of Clones: IL-2 Binding to Receptor Causes T Cell Proliferation
The release of IL-2 is the final signal in the transduction process that
initiates T-cell proliferation. Specific IL-2 receptors have been installed
on the surface of the T cell (details not shown). These receptors were
produced in response to TCR activation in a process very similar to what
the animation has shown for IL-2 production. IL-2 binds to these receptors,
causing the T cell to divide. The identical daughter T cells in turn will
produce more IL-2 and IL-2 receptors, stimulating their daughter cells
in turn to divide, producing thousands of identical T-cell clones in a
matter of hours. One T cell with specificity for one particular antigen
has now become an army of T cells, all specific for the same antigen.
These T-cell clones will activate B cells to produce the antibodies that
will ultimately destroy the invading microbe.
Summary of the action in Part 6:
- IL-2 protein binds to receptors on the surface of the T cell.
- The T cell proliferates.
- One T cell with specificity for one particular antigen has now become
an "army" of identical T cells.
T Cell Background
The immune system is our body's defense mechanism against foreign invaders
such as viruses and bacteria. Several types of cells and organs make up
the immune system. The specialized cells of the immune system, called
lymphocytes, are initially produced in the bone marrow. Lymphocytes mature
either in the bone marrow to become B cells or in the thymus to become
T cells. B cells produce antibodies that circulate in the blood or the
lymphatic system. Antibodies attach to foreign antigens and mark these
invaders for destruction by other cells in the immune system.
T cells have two major roles. They can become cytotoxic T cells capable
of destroying cells marked as foreign. Cytotoxic T cells have a unique
surface protein called CD8, thus they are often referred to as CD8+
T cells. Alternatively, T cells can become helper T cells, which work
to regulate and coordinate the immune system. Helper T cells have a unique
surface protein called CD4 and are thus often called CD4+
T cells. Helper T cells have several important roles in the immune system:
1) responding to activation by specific antigens by rapidly reproducing;
2) signaling B cells to produce antibodies; and 3) activating macrophages.
The animation focuses on the activation and proliferation of helper T
cells.
The immune system has evolved multiple mechanisms to distinguish foreign
antigens from self- antigens. One way in which T cells recognize foreign
antigens is through the major histocompatibility complex (MHC). For example,
a foreign invader such as a pneumococcal bacterium (which can cause pneumonia)
may be engulfed by a macrophage. The bacterium is processed in the macrophage
in such a way that only a portion of the bacterium is attached to the
cell surface of the macrophage. On the surface of the macrophage, this
bacterial antigen is "presented" and bound as part of the MHC. Many types
of MHC molecules exist; helper T cells specifically recognize antigens
of the MHCII type. The macrophage is just one cell type that may present
antigens in the MHCII complex; other cells, including B cells and dendritic
cells, may also act as antigen-presenting cells (APC).
Only a few helper T cells in the immune system will recognize the specific
antigen presented by the APC. One helper T cell must become thousands
of T cells to mount the immune response against the foreign invader. The
animation illustrates the passage of signals through the cell that results
in T-cell proliferation. The many resulting T cells can then stimulate
other T cells to attack the foreign pathogen, or they can stimulate B
cells to produce antibodies. These antibodies, along with other cells
in the immune system, then destroy the foreign invaders, helping to ensure
the health of the individual.
Resources
Books
Roitt, I., Brostoff, J., and Male, D. Immunology. 4th ed. London:
Mosby, 1996; pp. 4.1–6.12.
Howard Hughes Medical Institute. Arousing the Fury of the Immune System.
Chevy Chase, Md.: Howard Hughes Medical Institute, 1998.
Howard Hughes Medical Institute. Exploring the Biomedical Revolution
and classroom guide. Chevy Chase, Md.: Howard Hughes Medical Institute,
1999; distributed by Johns Hopkins University Press.
Web Resources
National Institute of Allergy and Infectious Diseases, National Institutes
of Health: The Immune System
An introduction to the immune system.
http://www.niaid.nih.gov/topics/immunesystem/Pages/default.aspx
Davidson College: Immunology Course Includes animations on MHCII antigen
loading and IP3 and Ca++
ion waves.
http://www.bio.davidson.edu/courses/Immunology/Bio307.html
Related HHMI Resources on BioInteractive.org
Rheumatoid Arthritis: Past, Present, and Future
A lecture by Andrew C. Chan, M.D., Ph.D.
http://www.hhmi.org/biointeractive/immunology/talks.html
Virtual Bacterial ID Lab
http://www.hhmi.org/biointeractive/vlabs/bacterial_id/
Virtual ELISA Lab
http://www.hhmi.org/biointeractive/vlabs/immunology/
Antibiotics Attack
http://www.hhmi.org/biointeractive/Antibiotics_Attack/frameset.html
Pathogenic E. coli Infection Mechanism (Infectious Diseases)
http://www.hhmi.org/biointeractive/disease/ecoli.html
Intracellular Infection by Salmonella (Infectious Diseases)
http://www.hhmi.org/biointeractive/disease/salmonella.html
References Used in Developing the Animation
Reviews
Bubeck-Wardenburg, J., Wong, J., Futterer, K., Pappu, R., Fu, C., Waksman,
G., and Chan A.C. 1999. Regulation of antigen receptor function by protein
tyrosine kinases. Prog. Biophys. Mol. Biol. 71 (3–4): 373-92.
Chan, A.C., and Shaw, A.S. 1996. Regulation of antigen receptor signal
transduction by protein tyrosine kinases. Curr. Opin. Immunol.
8 (3): 394–401.
Dustin, M.L., and Chan, A.C. 2000. Signaling takes shape in the immune
system. Cell 103 (2): 283–94.
Primary papers
Chan, A.C., Dalton, M., Johnson, R., Kong, G.H., Wang, T., Thoma, R.,
and Kurosaki, T. 1995. Activation of ZAP-70 kinase activity by phosphorylation
of tyrosine 493 is required for lymphocyte antigen receptor function.
EMBO J. 14 (11): 2499–508.
Kong, G., Dalton, M., Wardenburg, J.B., Straus, D., Kurosaki, T., and
Chan, A.C. 1996. Distinct tyrosine phosphorylation sites in ZAP-70 mediate
activation and negative regulation of antigen receptor function. Mol.
Cell Biol. 16 (9): 5026–35.
Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R.P., and Samelson,
L.E. 1992. LAT: the ZAP-70 tyrosine kinase substrate that links T cell
receptor to cellular activation. Cell 92 (1): 83–92.
Teaching Tips
Signal Transduction Cascades
1. Discuss the process that occurs in signal transduction cascades. In
the animation illustrating T-cell proliferation, signal transduction begins
when a foreign antigen carried by the antigen-presenting cell (APC) is
recognized by the T-cell receptor. Several molecules located in or near
the cell membrane mediate the transmission of signals, resulting in the
production of inositol triphosphate (IP3), which
travels into the cell's cytoplasm. IP3 initiates
the release of calcium, which, through several intermediate steps, allows
a transcription factor to enter the nucleus. Within the nucleus, this
transcription factor and other essential factors trigger the expression
of the gene interleukin-2 (IL-2). IL-2 protein is produced and released
extracellularly, where IL-2 binds to receptors on the T cell. This receptor/IL-2
protein complex signals the T cell to proliferate.
2. Compare the signal transduction cascade in the helper T cell with
that occurring in other cells in other biological processes. Discuss messenger
molecules that are used by many cell types, such as IP3
and Ca++. Consider showing other animations
that describe these processes in more detail.
Molecular Switches
1. Discuss activation of kinases by phosphorylation. Discuss how kinases
activate other kinases.
2. Discuss dephosphorylation by phosphatases. Does dephosphorylation
always inactivate a molecule? (No.) Discuss how in the animation, NF-AT
(nuclear factor of activated T cells) cannot pass through the nuclear
membrane in its phosphorylated state. However, when dephosphorylated,
it passes through the membrane and can initiate gene transcription of
IL-2.
3. Describe scenarios in other cascades in which phosphorylation activates
or deactivates different molecules.
4. What do these general regulatory principles tell us about molecular
switches? For example, there are many levels of control, pathways intersect,
and many molecules need to be activated (that is, switched "on") in order
for the molecule to do its job.
Docking Sites
1. Describe how the animation demonstrates that molecules have multiple
functions. For example, ZAP-70 (zeta-associated protein-70) both docks
at a site—ITAM (immunoreceptor tyrosine-based activation motif)—and
prepares another docking site—LAT (linker for activation of T cells).
2. Discuss the biological significance of molecules having multiple functions
in one cell.
3. Fill out the following chart to demonstrate how the two docking sites
in this animation are activated. Use the chart to illustrate the multiple
functions of ZAP-70.
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Activates the docking- site molecule
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Docking site
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Binds to docking site
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Completed Chart:
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Activates the docking-site molecule
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Docking site
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Binds to docking site
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Lck
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ITAM
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ZAP-70
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ZAP-70
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LAT
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PLCg
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Blocking Molecular Transduction Cascades with Drugs
1. Discuss how researchers use molecular pathways, such as those shown
in the animation, to design drugs for patients with autoimmune diseases
such as rheumatoid arthritis. Because each step in a signal transduction
cascade is dependent on the preceding step, such a cascade contains several
potential points at which drugs can be targeted to specifically interfere
with the progression of the cascade.
2. Discuss the molecular mechanisms of the drug cyclosporin in the context
of the animation. How does cyclosporin block the molecular signaling cascade?
Cyclosporin inhibits calcineurin. As a result, transcription factors normally
stimulated by calcineurin to turn on IL-2 production are blocked. IL-2
is not produced when cyclosporin is administered. Cyclosporin has been
used to block the immune response, particularly for patients with autoimmune
diseases in which the patient's immune system attacks its own cells.
3. Discuss how inhibiting ZAP-70 can block the pathway that would otherwise
lead to increases in Ca++. Discuss whether
drugs that target ZAP-70 may be able to reduce symptoms in autoimmune
diseases.
Collaborative Learning and Communication
Have students watch the animation in pairs. They should focus first on
the four major themes and then view actions and descriptions of specific
molecules. Provide students with some directed questions. Students can
also prepare presentations related to the animation. Topics might include
signal transduction cascades, molecular switches, assembly of molecules
at docking sites, or targets for drug design.
Credits
Director: Dennis Liu, Ph.D.
Scientific Direction: Andrew Chan, Ph.D.
Scientific Content: Donna Messersmith, Ph.D.
Animators: Chris Vargas, Eric Keller
Editorial review: Dean Trackman
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