Identify the cell type
Are T cells and B cells distinguishable microscopically?
They are distinguished by their surface molecules.
What are the two major types of T cell receptors (TCRs)?
In which type of lymphocyte might you find a CD3 molecule?
CD3 acts as a signal transduction molecule for the TCR.
Learning objective 1
List the important surface molecules that distinguish T and B cells and describe their function.
T cell receptor (never secreted)
- Two major categories: Alpha-beta and Gamma-delta
- CD4 receptor on Th cells: binds constant region of MHC II
- CD8 receptor on Tc cells: binds constant region of MHC I
- signal transduction molecule for TCR
B cell receptor (immunoglobulin with transmembrane region)
- Ig Alpha and Ig Beta act as signal transduction molecules
- Complement, Fc, and cytokine receptors.
What does CD stand for?
Cluster of Differentiation
CD terminology is used to describe molecules located on the cell surface.
What is the function of CD 11 and CD 18 molecules?
These are integrin adhesion molecules.
Describe the process of flow cytometry to classify cells by immunophenotype.
Antibodies (example: anti-CD8) covalently bound to dyes are added to cell populations. The antibodies will bind to their specific receptor and specific cell types (example: Tc cells) will glow specific colors.
The flow cytometry machine will then count the numbers of cells glowing different colors.
Describe the function of a fluoresence-activated cell sorter.
A machine that rapidly separates cells based on size or color.
Cells tagged with fluorescent antibodies can be assigned a charge and separated into different containers.
Learning objective 2
Explain how B and T cells are identified
T and B cells cannot be distinguished with a light microscope. They are distinguished through which surface molecules they express.
B cells express BCRs and the transduction molecules Ig Alpha and Ig Beta.
All T cells express CD3. Depending on T cell type, they may express CD4 or CD8.
T and B cells can be identified and counted through the process of flow cytometry. They can be separated into pure populations with a fluorescence-activated cell sorter.
Two theories exist for the generation of antibody diversity: the insructive theory and the clonal selection theory. Which is correct?
Clonal selection theory
B cells are not "taught" to make antibodies by being exposed to an antigen. Instead, each B cell has a specific B cell receptor. If that B cell happens to encounter its antigen, it will undergo the process of clonal expansion.
True or False:
All of the offspring of a single B or T cell have the same antigen specificity as the original cell.
When a B or T cell encounters its antigen, it will undergo mitosis to produce a number of daughter cells expressing the same antigen specificity. This process is termed clonal expansion.
What process is being illustrated here?
A lymphocyte has encountered its antigen and undergone mitosis to produce a number of daughter cells. Note how all daughter cells express the same antigen-specific receptor.
True or False:
During an infection with a specific pathogen, we will most likely see a monoclonal antibody population circulating in the plasma.
Pathogens have many epitopes. Thus, several different B cell populations will undergo clonal expansion, and the resulting antibody population will be polyclonal.
This process is termed polyclonal activation
When a B cell is activated, what 2 cell types are produced from its clonal expansion?
Memory B cell
What is the function of a plasma cell?
What is the function of a memory B cell?
If an antigen is encountered a second time, memory B cells will quickly differentiate into effector cells (i.e. plasma cells) for quick secretion of antibodies.
Describe what occurs when a B cell recognizes a self antigen during its maturation.
This process promotes Tolerance of self antigens.
Learning Objective 3
Explain the basic concepts of clonal selection theory and how it relates to tolerance and memory.
Lymphocytes carry receptors specific to a single antigen.
If a lymphocyte encounters a self antigen during development, it undergoes apoptosis. Tolerance
If a mature lymphocyte encounters its antigen, it will undergo clonal expansion into effector cells or memory cells. Memory cells are quickly activated during a secondary infection to promote a quick immune response. Memory
Where do B cells mature?
In the primary lymphoid tissues
Bone marrow in mammals
Bursa of Fabricius in birds
Which immunoglobulin serves as a maturation marker for B cells?
How do B cells develop their BCRs?
Rearrangement of their DNA
This occurs in the primary lymphoid tissues
Where do B cells typically encounter their antigens?
In the secondary lymphoid tissues
The antigen will drain into lymph nodes via the lymphatics, where a B cell can contact the antigen.
How long does it take for a B cell to differentiate into effector cells?
Why isn't IgM immediately present in the plasma after initial exposure to an antigen?
B cells are undergoing clonal expansion.
Recall that it takes approximately 5 days for B cells to differentiate into effector cells.
During an infection, an individual can experience enlarged lymph nodes. Why is this?
B and T cells are undergoing rapid clonal expansion.
This process can be painful.
During an initial infection with an antigen, some B cells will differentiate into plasma cells. Which antibody class will they secrete initially?
What influences a B cell to switch antibody isotypes?
A mixture of cytokines released from Th cells.
In this way, B cells can be influenced to release an antibody isotype more specific to the antigen in question (ex: switch from IgM to IgG).
Why is antibody production faster in the secondary response than in the primary response?
In the secondary response, memory cells are already present and can differentiate into plasma cells.
In the primary response, lymphocytes must undergo clonal expansion, which takes time.
Learning Objective 4
Compare and contrast a primary and secondary response. Include the basis for the difference and lag time, titer, antibody class, and antibody affinity.
- Lymphocytes encounter their antigen and undergo clonal expansion.
- After 5 days, B cells have differentiated into plasma cells and memory cells. Plasma cells begin releasing IgM.
- Based on infection type, Th cells can influence B cells to switch the isotype of antibody being released.
Secondary response (anamnestic response)
- Memory lymphocytes are already present in circulation and can differentiate into effector cells more rapidly.
- Some IgM is produced, similarly to primary response.
- Clonal memory cells produce the specific antibody isotype from the previous infection without having to class switch.
- Antibody production is higher titered and higher affinity.
Describe the primary differences between a B cell and a plasma cell.
B cells are predominantly nucleus and are not very active. Will circulate for 4 months searching for its antigen.
Plasma cells have a lot of RER and Golgi and are protein-exporting machines. Release about 300 antibody molecules per second. Some live 5-6 days and then die. Others are long-lived for periods of up to years.
Define "Central tolerance"
If a lymphocyte encounters a self antigen during maturation, the following events may occur:
- Rearrange DNA to edit BCR
CD4+ T cell
- Become a Treg cell, which suppresses T cell responses to the antigen
Define "peripheral tolerance"
If a mature naive lymphocyte encounters a self antigen and does not receive danger signals, it may:
- Die by apoptosis
- Become anergic (inactive)
- Be suppressed
A mature naive B cell will express which two immunoglobulins on its cell surface?
IgM and IgD
Recall that IgD is a marker of B cell maturation
Learning Objective 5
List the way tolerance is induced - include central and peripheral tolerance.
- If an immature B cell recognizes self, it undergoes apoptosis or rearranges its BCR.
- If an immature CD4+ T cell recognizes self, it becomes a Treg and suppresses T cell responses to that antigen.
- If a mature naive lymphocyte recognizes self without the presence of danger signals, it may undergo apoptosis, become anergic, or be suppressed.
True or False:
IgM is a monomer when acting as a BCR on the B cell surface.
Learning Objective 6
Understand the role of IgM and IgD in B cell development and where they are expressed.
1. Immature B cell rearranges its genes to develop its specific BCR in the form of an IgM molecule.
2. If IgM recognizes self in primary lymphoid tissue, cell will be deleted or will rearrange its genes again to form a new BCR.
3. B cell migrates to secondary lymphoid tissues, where it develops IgD expression. IgD is a B cell maturation marker.
4. B cell can now be activated by contacting its antigen.
Suppose you wanted to produce a large batch of monoclonal antibodies. How would you go about doing it?
Inject mouse with an antigen to produce clonal expansion in the spleen. Harvest the spleen for B cells.
Combine B cells with myeloma cells. This forms a "hybridoma," creating an immortal line of antibody-producing cells.
Dilute cells into assays containing 1 cell apiece. Test supernatant for desired antibody.
Harvest cells that are producing the desired antibody.
Why is it necessary to create a hybridoma when harvesting monoclonal antibodies instead of just growing B cells in vitro?
After a few divisions, B cell populations will die off.
Thus, it is necessary to combine them with immortal myeloma cell lines.
Learning Objective 7
Define hybridoma and include in your definition the cells that are used to create hybridomas and the purpose of hybridomas
Hybridoma: the combination of a B cell with a myeloma cell.
The B cell portion of the hybridoma produces antibody, while the myeloma (plasma tumor cell) confers immortality to the cell line.
This technique allows for the production of large batches of monoclonal antibodies.