Something that binds to an antibody
That part of the antigen that can bind to the antibody
An antigen that is capable of eliciting an immune response
A lymphocyte mediated immune response where all the activated lymphocytes are derived from a single clone
A lymphocyte mediated immune response where the active lymphocytes are derived from multiple clones
The binding of an antigen with an antibody or TCR, which was originally made specifically for another antigen. An example of this is rheumatic fever. A patient gets infected with streptococcus and develops antibodies for its antigen. It turns out that antigens of streptococcus are similar to myocardial antigens so these antibodies also bind to the antigens of the heart cells. This results in rheumatic fever
Does B cell needs MHC to bind to the antigen
No! They can bind to anything, like lipids, proteins, sacchardies etc
What is the structure of TCR and BCR
Compare the properties of TCR and BCR
Whats the difference in the binding pattern of TCR and BCR
TCR bind only to peptides that are sequential part of the protein, those peptides can be from any protein that can lie virtually anywhere on the cell.
BCR can bind to any kind of antigen, however that antigen has to be on the cell surface membrane. The antigen for BCR can be sequential or it can made from conformation of more than 1 peptide (or other) chains coming together
What are the properties of super antigens
They bind outside the MHC, they are only limited to MHC 2 and CD4 T cells. This leads to a polyclonal T cell response leading to a cytokine storm
What kind of BCRs are there and how are they made
There are 2 kinds of BCRs, one lie on the cell surface membrane and the other are solouble. These 2 varieites are arrived by the fact that a BCR is made of a variable domain, a constant domain and transmembrane domain. There are 2 polyA tails added, one before the trnx membrane domain and one after it. If the splicing is done such that the first polyA tail is used, then the trns membrane domain is chopped off, this leads to a solouble BCR whereas if the latter polyA tail is used transmembrane domain is kept and BCR is expressed on the surface of the B cell.
Explain the structure of an antibody in detail
1. There are variable and constant regions.
2. There are light chain and heavy chains
3. On the top of an antibody are the hypervariable region that makes physical contact with the antigen. These are called Complementarity Determining Regions (CDRs)
4. Fab fragment region, this region extends all over the variable region and into the constant region (these are the arms of the Y)
5. FC fragment region, this is the stem of the Y, this crystallizes when the antibody is digested.
What is an another important region of the antibody
Hinge region. It has alot of glycine residues that provide greater flexibility to the arms of the Y to attach to the antigen.
What happens downstream when an antibody binds to an antigen
The hinge region allows the antibody to tightly bind to the antigen which leads to conformation changes in the FAB region such that a torque directed in the FC region which leads to binding of the FC region with the antigen? I think.
What are the isotypes of antibodies
Antibodies are classified acoording to the isotypes of the heavy chains which gives rise to categoire IgG, IgE, IgM etc
Key facts to know about the isotypes
1. IgM is the heaviest/biggest
2. IgD and IgE are rare in serum, most is IgG
3. Half life of IgG is 3 weeks
What are the functions of these antibodies
What is a monomer and a polymer is antobodies, what is a polymer called?
What allows this polymerization
What antibodies form these polymers
Monomer is 2 light chains and 2 heavy chains whereas a polymer of an antibody would be more than 1 of this form of assembly, it is called a multimer.
This is a sequence of amino acids in the stem that are called the J chain that hinge the units of antibodies together
IgA and IgM are mutlimers
How many binding sites are present in a monomer antibody
What is affinity vs avidity
Affinity is the ability of the ligand to bind with the active site of a protein whereas avidity is the strength of protein-protein interaction when multiple binding sites are present
Explain the process of development of B/T cells
1. Everything starts with a Hematopoietic Stem Cell.
2. It differentiates into a common lymphoid progenitor.
3. This differentiates into pro B/T cells.
4. Differentiates into pre B/T cells that express one antigen receptors
5. Cell proliferation which further leads presentation of both of the antigen receptors.
6. The final step is selection, those with weak antigen recognition go on to become mature B/T cell whereas the ones with strong antigen recognition ability undergo apoptosis.
What are the goals of B cell development
1. Functional lymphocyte
2. Diversity - needs to recognize all forms of antigens
3. Limited self-reactivity
What does the signaling function of the BCR
Ig alpha and Ig beta, they have long cytoplasmic tails that does the signaling cascade function of the BCR
Explain the structure of the heavy chain
It consists of the variable regions and constant regions. The variable region can be further divided into gene segments which are:
Constant region is the same except for different isotypes of the BCR
How does genomic regarrangement of IgH gene take place
1. First D to J is connected
2. Then this segment is connected with one of the V regions
3. A group of enzyme mediate this combination, they are called V(D)J Recombinase. There are all kinds of enzymes involved in these such as DNA ligase and DNA helicase. Also the following enymes are involved that are only found in lymphocytes:
- Recombination Activating gene 1 and 2 (RAG 1 and 2), both are needed since they dimerize
- Terminal Deoxynucleotidyl Transferase (TdT)
Schematic diagram of genomic recombination of IgH locus
How many different kinds of B cells are there in a human body?
How is this diversity achieved?
Theoretically what is the maximum number of different B cells that there can be in a human body?
There are about 109. Combinatorial diversity only gets us 10^6, further diversity is achieved by junctional diversity where nucleotides are randomly added or removed at the junctions where V, D and J meet. This is done by TdT and this gets the diversity up to 10^9.
Theoretically B cells can in 10^11 number.
What is a pre B cell
This is when only one of the antigen receptor is expressed. This antigen receptor is the heavy chain. Heavy chain expressed by itself will not stay on the cell surface membrane since it needs a light chain, so a surrogate light chain is expressed called the Vpre-B and lamda 5.
Also pro B cell express Ig alpha and Ig beta.
What does the pre-BCR induces
3. Allelic exclusion
5. Light chain rearrangment
What is the idea of allelic exclusion.
Why is this necessary?
Genomic recombination is a highly error prone process since we are rearranging the pieces of DNA. This recombination continues on both alleles until successful recombination is achieved or the cell runs out of coding sequence.
Once successful genomic recombination is done, this finished recombination inhibits the rearrangement of the other allele. This phenomena is called allelic exclusion.
This is necessary in order to ensure that a given B cell only expresses one BCR at a time!
How does progress from pre-BCR to BCR takes place
1. VJ recombination
2. Re-espression of RAG 1 and 2 (bit NOT TdT)
What is present in the gene that codes for the light chain.
What are the types of light chain
1. There are Variable regions.
2. There are Joining regions.
3. Diversity regions are absent
4. There are 2 types of: kappa and lamda
Explain the process of selection for B cells
There is positive selection which basically is a green light for that B cell to advance to next stage.
In negative selection either the BCR doesn't work or it binds to the self antigens. If either of these are present then the B cell can undergo BCR editting or there can be clonal deletion
Explain the process of BCR editting and clonal deletion
If the BCR protein doesnt work or if it self reactive then the light chain (which is the last step of the process of BCR development) undergoes further recombination to make a light chain such that BCR wouldnt be self reactive.
This can be done several times. If the B cells succeeds, they undergo positive selection and emerge from bone marrow as naive mature B cells. If the B cell does not succeed, it undergoes clonal deletion which basically is apoptosis.
What does mature B cells express
IgD and IgM
Explain the process of B cell development again
2. Common Lymphocyte Progenitor
3. Pro B cell - Express RAG 1, RAG 2 and TdT
4. Pre B cell, by defintion this expresses heavy chain. These then reexpress RAG 1 and RAG 2
5. Immature B cell - express a rearranged heavy chain and the light chain, these cells are subject to selection
6. Mature naive B cell - the cell that undergoes positive selection
What are the goals of T cell development
1. Functional lymphocytes
2. Diverse Repertoire
3. Limited self reactivity
What is the first step in the development of a TCR
Generation of pre-TCR
What is analogous in the development of TCR and BCR
In pre-BCR development, we made the heavy chain first. In pre-TCR development we will make the beta chain first.
How is pre-TCR made
1. Recombination of the D and J segments
2. Then V to DJ is added to form VDG
Same enzymes are used RAG1, RAG2 and TdT which are only found in lymphocytes and then some of the more general enzymes such as DNA ligase and helicase etc.
If a patient is deficient in either RAG1 or 2 or TdT they will have a deficiency of both T cell and B cells
Explain the concept of TRECs
T cell Receptor Excision circles (TRECs) are the circular loops of DNA that form during the process of genomic recombination to generate VDG. These circular DNAs are not degraded in the T cells (and the B cells) for a long period of time. TRECs can be measured quantitatively by quantitative PCR where extracts from thymus can be taken and TRECs can be quantified. This is done in newborn screening so if a child has low TRECs the child will be deficient in T and B cells and they will need a Stem Cell transplant.
Quick question: what is missing from the TCR alpha chain
Diversity region, just like they are missing in BCR light chain
Explain the process of development of TCRs
1. Originate from the bone marrow, from HSC and then into CLP
2. Develop pre-TCR by making the beta chain first
3. Move to thymus from bone marrow, these cells are now called DN thymocytes (double negative). The first decision the cell makes is that whether it wants to be alpha-beta or gamma-delta T cell.
4. Now they become DP thymocytes, expressing both the CD4 and CD8 receptors. This step of the process is analogous to selection of BCR
5. These cells mature into SP thymocytes (single positives). They express TCR alpha-beta and CD4 or CD8
Important: We should be able to tell by the structure of MHC whether they are class I or class II
Explain the process of selection for TCR
DP thymocytes that have TCR that can detect MHC complexes are positively selected to become CD4/CD8 cells, DPs that have TCR that cannot detect MHC undergo apoptosis.
If the DP recognize class 1 they become CD8 cells and if they recognize class 2 they become CD 4 cells
Explain negative selection
TCRs that have self reactivity upon interacting with MHC undergo apoptosis
What is the dilemma of selection in TCR
TCRs have to interact with MHC to undergo positive selection but at the same time they cannot have self reactivity. So the question is how can interactions with MHC lead to both positive and negative selection.
The answer to this is affinity model of thymic selection, if the MHC is recognized by weak affinity those T cells undergo positive selection, T cells that bind with high affinity undergo apoptosis by negative selection
What are the other T cells and how are they selected
NKT cells, they detect glycolipids that are presented on CD1. CD1 is an MHC like molecule, its crytal structure looks just like MHC but it is not polymorphic like MHC.
What is important to remember regarding the NKT cells
There is a very limited repertore of NKT cells
What does the gamma delta T cells recognize
Small molecules like vitamins without the presence of MHC, they dont require MHC presentation of these molecules so they can identify solouble molecules as well as those present on the surface of the microbes
Explain the structure and role of thymus on T cell development
Thymus has thymic epithelial cells that express both MHC 1 and 2. Pre T cells enter the thymus at the cortex-medullary junction where they move to the cortex, become DP cells and then go into the medulla to become SP cells which then mature to become CD4/8 cells.
In the medulla are the dendritic cells influencing selection and presenting antigens.
Histology of thymus
Thymus is divided into lobes by trebeculae. Light staining region is the medulla
What do the arrows point to
Thymus with age. Adipose tissue, connective tissue and clusters of lymphocytes
What is flow cytometery
What is CD3
It is part of the TCR complex, it is expressed when the beta and alpha chain both are rearranged and expressed. CD3 expression increases as the T cells mature and express more TCR on the cell surface membrane
It is important to know the difference in faulty development of T cells that has lead to cancer in Pateint 1 and 2.
The top patient graph are graphs for peripheral blood smear (which should only have CD4 and 8) whereas the extreme left graph is a normal observed graph of thymus (which has all kinds of T cells)