6 - B Lymphocytes and humoral immunity Flashcards
Hyper-IgM syndrome is an immunodeficiency. What laboratory values can confirm this diagnosis?
Elevated levels of IgM
Reduced levels of IgG, IgA and IgE
What is the patient with hyper-IgM syndrome lacking?
Lacking the ability to …
- Undergo isotope switching
- A biological mechanism that changes a B cell’s production of immunoglobulin (antibodies) from one class to another, such as from the isotype IgM to the isotype IgG
- Produce antigen-activated B lymphocytes
What might a patient with hyper-IgM syndrome present with?
Case study
- Upper and lower bacterial respiratory infections
- Diarrhea caused by a common intestinal parasite
What is the treatment for a hyper-IgM syndrome?
Lifetime administration of intravenous immunoglobulin derived from pooled donor serum in order to reduce the number of infections
What is the prognosis for a patient with hyper-IgM syndrome?
Not great… 25% of all patients with this immunodeficiency will die from opportunistic infections before the age of 25 :(
What are B lymphocytes?
B lymphocytes are a subset of lymphocytes that are responsible for humoral immunity
- One of the three lymphocyte types
- 8-10 um in diameter
- Can’t tell the difference between T and B microscopically
- Named after birds - “bursa of fabricus”
What are B lymphocytes a precursor of?
B lymphocytes are a precursor of plasma cells
***B lymphocytes → develop → plasma cells***
When a B lymphocyte is not stimulated by an antigen, what is found on the surface? Why?
How many different antigens can each B cell bind to?
ONE!
Each B cell has ONE antigen specificity
What are B cell clones? How many antigens are they reactive to?
B cell clones are reactive to >109 antigens exist in the body at all times, without the need for exposure to these antigens
- For example, every individual currently has B lymphocyte clones specific for Ebola virus, HIV and Rabies virus even if never exposed to the viruses
- This is due to the random genetic recombination events that take place to produce B lymphocytes of >109 distinct antigenic specificities
What is a primary immune response?
The first time an individual encounters an antigen
- It takes 7-10 days to mount enough antibody production to fight it off
What is a secondary immune response?
A “memory” response, meaning that it is not the first time that the individual has encountered this antigen and they already have antibodies for the antigen in their immune system
- This will result in a fast immue response than a primary response
- Only 2-3 days
- Results in an overall higher level of antibody
What is affinity maturation? Does a primary or secondary response demonstrate affinity maturation?
The process by which B cells produce antibodies with increased affinity for antigen during the course of an immune response → SECONDARY immune response only
What are the stages of maturation of a B lymphocyte?
- Stem cell (from bone marrow)
- Pre-B cell (non-antigen responsive)
- Immature B cell (IgM specificity is developed, still in BM)
- Mature B cell (IgM and IgD are specific for same antigen)
- Activated B cell (stimulated by antigen)
- Plasma cell or Memory B cell
What is characteristic of a mature B cell?
Mature B cell
- Membrane IgM and IgD are specific for the SAME antigen
- They are responsive to antigen
What is a plasma cell? Where are they found?
Plasma cell
- The final stage of B lymphocyte maturation, characterized by the secretion of one antibody isotype
- Plasma cells are found in lymphoid organs and bone marrow
Describe the morphology of plasma cells
Morphology:
- Elongated cell
- Eccentric nucleus
- Perinuclear halo
- Abundant cytoplasm
See slide 9 for image
What is the specific job of a plasma cell?
Plasma cells
- Terminally differentiated antibody-producing machines
What is the lifespan of a plasma cell?
A few days, however some are very long-lived plasma cells that reside in the bone marrow
What two cell types must cooperate in order to produce antibodies in response to an antigen?
B cells and T cells
- Usually B and T cells each respond to a different epitope on the same antigen
- This interaction is class II MHC-restricted
What are MHCs againt?
Major histocompatibility complex (MHC)
- A set of cell surface molecules encoded by a large gene family which controls a major part of the immune system
- The major function of MHCs are to bind to peptide fragments derived from pathogens and display them on the cell surface for recognition by the appropriate T-cells
Describe the process of B cell antigen presentation
B cell antigen presentation
- The antigen is bound on the surface by the antibody
- The antigen undergoes receptor-mediated endocytosis
- The antigen is destroyed by the lysosomes
Note that this is the process of class II MHC
How long does B cell antigen presentation take?
1-6 hours
What is the effect of having fewer lysosomes?
Less efficient processing of antigens
What happens when a non-protein antigen is presented?
It can’t be complexed to MHC protiens
Describe the interactions between B and T lymphocytes during antigen presentation
Antigen presentation
- The antigen activates BOTH cells
- This drives antibody production
- There are two sets of interactions
- B7 (B cell) interacts wtih CD28 (T cell)
- CD40 (B cell) interacts with CD40L (T cell)
What is the role of accessory proteins in B cell antigen presentation?
Accessory proteins will interact with class II MHC-bound peptides, which results in…
- T cells releasing cytokines
- B cells becoming activated
- Plasma cells producing antibodies
What is the outcome of a B cell activation?
A cascade of events
- Neutralization of microbes
- Opsonization and phagocytosis of microbes
- Antibody-dependent cellular cytotoxicity
- Complement activation
- Phagocytosis of microbes opsonized with complement fragments
- Inflammation
- Lysis of microbes
What does it mean that protein antigens are “thymus-dependent”?
Protein antigens require the assistance of T cells - these come from the thymus, making protein antigens thymus-dependent
Why are non-protein antigens “thymus-independent”? (TI)
Non-protein antigens are “thymus-independent” because they dont’ need T cells for processing
- All the necessary cell signals can be provided from B cells (made in bone marrow, not in the thymus)
- Examples of nonprotein antigens include polysaccharides, nucleic acids and lipids (which are components of many bacteria)
There are two classes of TI antigens… TI-1 and TI-2. What constitutes a TI-1 antigen? What is an example of a TI-1 antigen?
TI-1 antigens
- Bind non-immunoglobulin receptors and promote polyclonal expansion
- Best example: Lipopolysaccharide (LPS) - acts as a B cell mitogen
What constitutes a TI-1 antigen? What is an example of a TI-1 antigen?
TI-2 antigens:
- Bind through B cell surface immunoglobulin (only activate antigen-specific B cells)
- E.g. - Polysaccharide antigens with repeated epitopes
For TI antigens, explain the memory cells, isotope switching and affinity maturation processes
With all TI antigens → no memory cells, no isotype switching, no affinity maturation
What components of a B cell help in signal transduction? Why is this necessary?
The cytoplasmic tails of the antibody are not long enought to transduce signals by themselves
- Ig-alpha and Ig-beta accomplish the signal transduction
- This serves the same function as T cell CD3 proteins
What is Ig gene rearrangement? What is this theory believed to be true?
We know that each antibody is specific to one antigen and that our body has many of these… It is not realistic to think that we have a separate gene for each antigen because:
- Too much of the cell’s genome would need to be used to encode antigen-specific antibodies if each specificity was encoded for by a single gene
- Antibodies have a shared constant regions, but have a greatly differing variable region... The “one gene-one protein” theory doesn’t account for the similarities
- The rearrangement theory means that there are multiple gene segments that encode a single protein
What are the structural components of an antibody?
Antibodies
- 2 heavy chains
- 2 kappa or 2 lambda light chains
Who postulated the gene rearrangement theory of Ig diversity?
Dreyer/Bennett in 1965
Heavy and light chains are encoded by gene segments on different chromosomes… Where are these located
Gene locations
- 5’ end - V (variable) region of gene segments
- Heavy chain D (diversity) region is after V
- J (joining) region is after V and D
- 3’ end - C (constant) region of gene segments
What gene segments does the heavy chain encode? What does the light chain encode?
Heavy chain: V, D, J
Light chain: V, J
Remember: C is not in here because it is always constant, think of heavy is having more, it is heavier, and the extra one is “D = dense” making it the heavy chain
Sooo… How does this generate diversity? What are the molecular mechanisms?
Generating diversity
- Pairing of heavy and light chains
- Combinatorial diversity
- Junctional diversity
- Somatic mutation
What is combinatorial diversity?
Combinatorial diversity: different gene segments rearranged
What is junctional diversity?
Junctional diversity: nucleotide addition/removal occurs at joints between segments (i.e. by using TdT)
How does a somatic mutation create diversity?
Somatic mutation: point mutations in variable regions of heavy and light chains
What types of cells have immunoglobulin genes?
How do B cells selectively rearrange gene segments in teh bone marrow?
- “Recombinases”
- Loops out
- Excises DNA
- Ligates the strand back together
- The “recombinases” are then coded as either RAG1 or RAG2 genes
- Mutations in this gene can cause SCID (severe combined immunodeficiency)
What is the importance of gene rearrangement?
Importance of gene rearrangement:
- Small number of gene segments → many different proteins
- Random selection of gene segments → unique antigen receptors
- DNA is irreversibly lost during recombination
- Each progeny of a B cell will inherit the same receptor specificity
What is the process of heavy chain rearrangement? When does it occur?
Heavy chain rearrangement happens first
- Random D and J segments brought together, intervening DNA deleted
- V gene segment randomly selected and placed 5’ to the DJ segment to form VDJ segment
- Intron separates VDJ segment from C region gene Cm and Cd (which remain in the 1o RNA transcript)
- Processing of 1o RNA → splice out introns, remove Cd, add poly-A tail
- mRNA → cytoplasmic m-heavy chain (pre-B cell stage)
What happens if the rearrangement on the first heavy chain gene is not successful?
The heavy chain gene on the other chromosome is rearranged… You inherit two sets of heavy chain genes, making this possible
What is alleleic exclusion?
Allelic exclusion:
- If the first rearrangement is productive, then the genes on the other chromosomes won’t be rearranged
What happens if the first attempt and the second attempt (allelic exclusion) to rearrange the heavy chain genes are unproductive?
Cell death
How many light chain genes do we inherit?
Four
- 2 kappa
- 2 lambda
Describe the process of light chain gene rearrangement
Light chain gene rearrangement
- A kappa gene will rearrange first
- If successful, allelic exclusion prevents the other light chain genes from rearranging
- If not productive, then the other kappa gene will attempt rearrangement, and so on until a productive rearrangement
What happens if all light chain rearrangemtns are not successful?
Cell death
What two things does an antigen-naive B lymphocyte express?
IgM and IgD
How do we asure that both IgM and IgD are both specific for the same antigen?
Alternative mRNA splicing
- Both are made from the same mRNA, just spliced differently
- This means that they have the SAME VDJ region
What happens when B cells are stimulated by an antigen?
The B cell shifts from a membrane antibody to a secreted antibody
- This involved differential splicing of the primary RNA transcript
- The primary RNA transcript codes for membrane-anchored and secreted forms, meaning that it just has to be spliced differently in order express the secreted form instead of the membrane-anchored form
- Membrane-anchoring sequences are spliced out and the result is the secreted form
What is isotype switching? When would this occur?
Upon stimulation by antigen…
- IgM and IgD-expressing B lymphocytes can switch to producing a different class/isotype of antibody
- IgG, IgM, IgA or IgE instead
There are two types of isotype switching. What are they?
- Long primary RNA transcript containing VDJ regions and many or all CH sequences produced, followed by splicing out of unneeded CH regions
- Deletion of intervening CH region genes (i.e. DNA) to align VDJ regions with a new CH gene. Uses “switch regions“ between CH region gene segments. IgM and IgD CH’s are deleted
During isotype switching, how is the new isotype selected?
It is dependent on T cell cytokines
- Example: if IL-4 is produced, IgE will be the isotype
When will a class of light chain be produced during isotype switching?
ALWAYS
- Isotype switching will always produce one class of light chain
- This means no switching
Will antibodies have a higher affinity for the antigen during a primary or secondary immune response?
Secondary!
How else can an increased affinity be achieved?
Somatic mutation in the V region of genes
Memory B cells express a higher affinity antigen receptor… What does this mean for a secondary response?
A smaller amount of antigen is needed to induce the secondary immune response compared to how much antigen it would have taken during the primary response
How are lymphocytes expressing the highest affinity of antibodies selected for?
The lymphocytes that are selected must…
- Be able to bind antigen
- Be stimulated by follicular helper T cells
This means that the highest affinity B cells will bind to antigens, interact with follicular hlper T cells, and produce high affinity antibodies
What happens to B lymphocytes with a low affinity for antigens or due to mutation, have no affinity?
Die by apoptosis
What are cell clones?
Descendents of one or a few B cells
