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Cloning an Army of T Cells for Immune Defense

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

  1. The T cell and APC come into contact.
  2. The MHCII-antigen complex and TCR bind.
  3. 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

  1. ZAP-70 contacts ITAM but fails to bind because ITAM is not activated (phosphorylated
  2. The plasma membrane reorganizes, bringing Lck, associated with CD4, close to ITAM. Lck phosphorylates ITAM.
  3. Phosphorylated ITAM region serves as a docking site for ZAP-70.
  4. ZAP-70 binds to phosphorylated ITAM.
  5. Lck phosphorylates ZAP-70.
  6. 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

  1. PLCg fails to dock to LAT because no phosphorylated tyrosine residues are present.
  2. Activated ZAP-70 phosphorylates tyrosine (circle) in LAT that is closest to membrane.
  3. PLCg docks onto phosphorylated tyrosine in LAT.
  4. PLCg is phosphorylated and activated.
  5. PIP2 within the T-cell membrane is catalyzed by activated PLCg into IP3 molecules.
  6. 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:

  1. IP3 binds to specific receptors on the ER.
  2. Ca++ is released from the ER in waves that travel throughout the cytoplasm.
  3. Ca++ forms a complex with calmodulin and calcineurin.
  4. The transcription factor NF-AT is unable to enter the nucleus when in a phosphorylated form (white circle).
  5. The Ca++/calmodulin/calcineurin complex dephosphorylates NF-AT.
  6. 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:

  1. NF-AT and other transcription factors activate transcription of the IL-2 gene.
  2. IL-2 mRNA is produced and exported out of the nucleus.
  3. Ribosomes translate the IL-2 mRNA into protein.
  4. IL-2 protein is processed in the ER and Golgi.
  5. 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:

  1. IL-2 protein binds to receptors on the surface of the T cell.
  2. The T cell proliferates.
  3. 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.

Activates the docking- site molecule

Docking site

Binds to docking site

Completed Chart:

Activates the docking-site molecule

Docking site

Binds to docking site

Lck

ITAM

ZAP-70

ZAP-70

LAT

PLCg

 

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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