Prelim 2 Flashcards
What does the adaptive immune system recognize in a virus like SARS-CoV-2?
The adaptive immune system recognizes an antigen. The S protein (spike) is a large antigen. It has MANY epitopes.
RBD
Receptor binding domain. This is the part of a protein/virus that helps it bind to host receptors
Epitope
Part of an antigen that an antibody binds
What cells make up the adaptive immune system?
The B cells and T cells which have antigen receptors
- antibodies are a secreted form of a B cell receptor
Antigen Receptors
- Receptor that binds to an antigen
- only 3 types: B cell receptors, antibodies, and T cell receptors. All used by the adaptive arm.
B Cell Arm
- One B cell expresses thousands of identical B cell receptors (Every BCR on that B cell is specific for the same antigen)
- Therefore, one B cell recognizes and responds to only one antigen. This B cell is binding a multivalent (repeating) antigen on the bacterium
- When B cells become plasma cells, they can secrete antibodies
B cell receptor
Expressed on the cell surface of a B cell. Part of a signaling receptor that instructs the cell.
Antibody
The only secreted antigen receptor, it is a form of immunoglobulin that is secreted by plasma cells and plasmablasts
*same rule of specificity applies to anitbodies
T Cell Arm
- One T cell expresses thousands of identical T cell receptors (TCRs) . Every TCR in that T cell is specific for the same antigen. A peptide is held by a specific MHC molecule on a host cell
- Therefore, one T cell recognizes and responds to one antigen.
- There is NO secreted form of a TCR
Which receptors are the immunoglobulins?
- BCR
- Antibodies
Antigen
- A molecule that is bound by an antigen receptor
- Can be from a threat or a non-threat
- Can be from the host (self) or non-self, pretty much any organic molecule
Immunogen
An antigen that induces an immune response
Draw the functional regions of the antigen receptors
- Surface immunoglobulin or B-cell receptor: antigen-binding site, light chain, heavy chain, transmembrane region
- Antibody: heavy chain, light chain, antigen binding site
- TCR: alpha chain and beta chain, antigen-binding site
- the variable regions are the antigen binding sites
Immunoglobulin structure
- The Fab portion (Top) is the amino terminus that binds the antigen
- The Fc portion (Bottom) is the carboxyl terminus. It is practically constant. It does not bind antigen, the Fc portion of Ab interacts with immune cell receptors and often, complement
- Fc portion can also be labeled with fluorophores & enzymes for experimental reagents
What do constant Fc regions do?
They give functions to antibodies (isotypes). For instance all IgM antibodies have the same Fc portion, but it is longer than the IgD Fc portion making them different.
Name the different isotypes
- IgM
- IgD
- IgE
- IgG
- IgA
IgM
- Made 1st to fix complement and form immune complexes. Very inflammatory
IgD
- At mucosal barriers, not understood
IgG
- Best all-round player: with 4 sub-isotypes. Most are inflammatory
- Useful tools in experiments and medicine
IgE
- For expelling large parasites and many allergy symptoms
IgA
- Dimeric form protects without inflammation
- Monomeric form is inflammatory, like IgG
Immunoglobulin Isotype
The ‘class; is defined by the heavy chains Fc constant region. The region interacts with the immune system to give the Ab different function. It is specifically encoded by the heavy chain C-region gene segments
* name the c regions
What antibody does the body start producing first and how does it switch?
- It starts by creating IgM and IgD. Through recombination of genes and switching, it can create IgG, IgE, and IgA
How is IgM secreted?
IgM is always secreted as a pentamer (5 identical Ab with 10 identical anitgen-binding sites). This increases avidity for an antigen.
Avidity
The overall strength of binding an Ab with multiple binding sites to an antigen in contrast to the affinity, which is the strength of binding at a single antigen-binding site
(ex. IgM doesn’t bind as tightly but it binds a bunch which increases avidity)
avidity is strength due to the amount of binding you are making whereas affinity is the strength due to the specificity of one antibody to an antigen.
Dimeric form of IgA
Is secreted across mucous membranes as 2 identical Ab connected by a J chain.
ex. In the breastmilk or intestines. Dimeric IgA is transported into the gut lumen through the epithelial cells and the IgA binds to the layer of mucus overlaying the gut epithelium
Can there be two different epitopes on the same antigen?
Yes! Oftentimes, two different antibodies can bind to two different epitotes on the same antigen
Linear epitope (draw it)
Is a continuous segment, like on the folded protein (i.e denatured antigen or sequential amino acids are the epitope)
Discontinuous epitope
Has discontinuous segments that require antigen to fold to form the epitope (the native configuration)
Multivalent antigens
Antigens have multiple epitopes that may be the same one repeated or be different
Bivalent antibodies
Have two identical antigen-binding sites
Complementarity-determining regions (CDR)
- The amino acid sequences of antigen-binding sites vary most in the hypervariable regions that encode loops. These loops actually bind the epitope
- The folded structure projects 3 loops. It is easy for the loops to bind an epitope
How many loops does each chain contribute?
The Ag-binding site has two chains (light and heavy). Each chain contributes 3 hypervariable loops. So, there are 6 CDRs that form 1 Ag-binding site. Which means there are 12 per antigen.
How is the strength of CDRs determined?
The 6 CDRs ( per 1 Ag-binding site) have a combined strength of binding that measured as affinity (Kd) for Ag
The function of BCR to activate B cells
- A B cell expresses BCR with identical specificity
- Some bacterium express a repeated antigen – BCRs that bind the Ags cluster close to one another
- Clustering brings internal signaling domains together
- The activities of signaling kinases start signals to activate the B cell
This is occuring on one B cell
Activation of B Cell Process
- BCRs that bind repeated antigens cluster close to one another which allows receptor-associated kinases to phosphorylate the ITAMs
- Blk, Fyn, and Lyn can bind to phosphorylated ITAMs and phosphorylate them again
- Syk binds to the doubly phosphorylated ITAMs, is activated, and sends activating signals to change gene expression in the nucleus
Agglutination
- Antibodies that bind 2 soluble antigens can act as arms and gather antigens (or the pathogens that express the antigens) into clumps (immune complexes)
- could be a toxin too
- Agglutination makes it difficult for pathogens to spread through a host
- The clumps are very inflammatory - Ab can fix complement and recruit phagocytes
What are the 2 general functions of antibodies?
- Neutralize infection or toxin
- Tag for elimination (many ways; all rely on the Fc portion of the Ab)
What is Ig-beta and Ig-alpha
They are the same on all B cells. They don’t bind antigens. They transmit intracellular signals
How many antigen binding sites are on a T cell receptor
1
How many antigen binding sites are on a B cell receptor
2
V and C part of the Alpha and Beta chains
- this is also present in the Fab of the antibody
- V i the variable part of the chain
- C is the constant part of the chain
Does a T cell receptor have signaling chains too?
Yes, like. BCR, its function is to transmit a signal to the cell
- TCR recignizes an antigen and then the CD3 chains transmit activation signals via ITAM regions
ITAM (for TCRs)
Sequence on signaling portions of CD3 chains that will be phosphorylated by signaling kinases
TCR Complex
Binding and signaling complex that is used as a receptor to activate T cell. Includes TCR and non variant CD3 chains
BCR Complex
Binding and signaling complex that is used to activate a B cell. Includes BCR and nonvariant Ig-alpha and Ig-Beta
How are TCR CDRs different than B Cell CDRs?
- T cell receptors have hypervariable loops that binds antigenic peptide AND MHC
TCR complexes signal alongside ___
Co-receptors. CD8 and CD4
- CD8 is associated with MHC class I
- CD4 is associated with MHC class II
The co-receptors bind to conserved regions of the MHC molecules
CD8+ T Cells
Killer T cells kill infected target cells (involves MHC I)
CD4+ T Cells
Helper T cells instruct other cells using cytokines (involves MHC II) –> ex. telling a macrophage to increase its killing capacity or differentiating a B cell into a plasma cell
Do B cells communicate with the host cell?
No, they do not. They can bind an antigen and activate signals to change its gene expression. Antibodies can also neutralize antigens. Only T cells are in communication with host cells.
CD “cluster of differentiation”
- Part of a numerical designation for many immune molecules. There are over 400 CD designations.
ex. CD8 is a coreceptor that binds to the MHC class I. T cells that express this are CD8 T cells which have certain properties like being cytotoxic. CD8 can be identified using anti-CD8 monoclonal antibodies
Do immunoglobulins and TCRs recognize the same antigens?
no
What do immunoglobulins recognize?
- Practically any organic molecule in any confirmation
- Can agglutinate antigens and opsonized antigens
- Epitopes can have many shapes
- Most epitopes are exposed on the surfaces of extracellular molecules or microbes
What do TCRs recognize?
- Only LINEAR peptide sequences and MHC that aren’t necessarily exposed on the surface of a protein
- They don’t agglutinate or opsonize
BCRs bind ____, ______ _____ that are in the _____ environment. They work to make ___ eliminate ____ threats.
BCRs bind intact, folded antigens that are in the extracellular environment. They work to make antibodies eliminate extracellular threats.
TCRs only bind _____ ___ ______ that are shown to them by ___ cells. They control _______ threats
TCRs only bind processed peptide antigens that are shown to them by host cells. They control cellular-associated threats
How can the Fc portion of antibodies be used for experimental purposes?
The antibody Fc portion can be labeled with something to detect when an antibody binds to an antigen such as fluorescent molecules or enzymes. Fluorescent molecules will glow in UV light and enzymes will produce color changes in samples
What are the three ways that antibodies can identify cells/antigens?
- Antibodies can identify cells floating in a solution (flow-cytometry)
- Antibodies can identify cells in tissue samples (immunofluorescence microscopy)
- Antibodies can identify molecules on test membranes in diagnostic tests
Flow cytometry
Identifies cell markers like CD3 chains in T cells and NKT cells, IgM on some B cells, and CD62L (naive T cells and naive B cells )
Immunofluorescence Microscopy
- You can attach a fluorophore to an antibody and then put them on samples. You can shine a specific wavelength (excitation) onto the sample, the fluorophore absorbs the sample. absorbs the energy, and becomes excited. Shortly after, it emits light at a longer wavelength which is the emission or on the visible spectrum
Diagnostic Tests
Antibodies can identify molecules on test molecules. For example, for a western blot you have have antigens of interest. You can have an antigen and have the primary antibody bind the signaling kinase antigen. Then, you can have a secondary antibody that is anti-IgG (which is the first antibody) so it binds the Fc conserved portion of the primary antibody.
- It will light up because the secondary antibody is enzyme linked so when you add a substrate, it will cleave it and the color will change.
Are antibodies commercially available?
Yes
Fluorophore
A fluorescent molecule that emits light when it is excited by high-energy light (i.e. Fluroescein (FITC), R-phycoerythrin (PE) can be covalently attached to Fc portions of Abs without interfering with Ab binding to the antigen.
What are the normal ranges for blood leukocytes
Neutrophiles: 40-75%
Monocytes: 2-10%
Eosinophiles: 1-6%
Basophiles: 0-1%
Total lymphocytes: 20-50%
B Cells: 30-40%
T Cells: 60-85%
NK Cells: 4-26%
What are the lymphocytes you need to remember?
B cells
T cells
NK cells
FSC
Forward scatter measures the size of the cell by sending a light through and seeing how large the scattering of light behind it is
Large cells?
Monocytes
Macrophages
Large granulocytes
Medium cells?
Neutrophiles
Small cells?
Lymphocytes
SSC
Side scatter measures the granularity of a cell or how complex it is (how many organelles it has inside)
Which cells have the highest granularity?
-Granulocytes (neutrophiles, NK cells, basophils, eosinophils)
Which cells have medium granularity?
Monocytes and macrophages
Which cells have the lowest granularity?
Lymphocytes
Steps of flow cytometry
- Incubate fluorophore-conjugated antibodies with a tube of cells. This is called ‘staining’ the cells with Ab. Wash away all Ab that aren’t bound to cells. Also put a fluorescent dye in tube that seeps into dead cells to stain them
- Pass the cells through the flow cytometer. Lasers excite the fluorophores. Detectors collect data on fluorescence and light scattering properties of each cell as it passes.
ELISA
ELISA measures antigen in a solution.
“Traditional” ELISA
- Coat the plate with sample
- Add the antibodies that bind the antigen. They have a enzyme
- Wash unbound antibody
- Add enzyme to make colored substrate `
Sandwich ELISA
- Coat the plate with antibody
- Add your sample in and it will bind with the antibody that is already on the plate
- Add a second antibody that will bind to a different epitope. This antibody has an enzyme
- Then add a substrate for the antibody with the enzyme and it will release the color into the solution
Why do you need a standard for ELISAs
You need a standard in ELISAs that actually has your antigen so you can compare it to the results that you do have.
Monoclonal Antibodies
Antibodies that are injected to bind a target (microbe or immune component) can treat or prevent disease
Give the example from class on a mAb and how it is used
- A therapeutic mAb could be Rituximab, an anti-CD20 IgG
- The tumor cell antigen could be CD20 if this were a B cell tumor (CD20 is expressed on all B cells)
- You could have NK cell killing of tumor cell by antibody dependent cell-mediated cytotoxicity (as well as a number of other ways due to tagging)
What are examples of other B cell targeted mAbs?
- Blinatumomab : CD19
- Rituximab : CD20
- Alemtuzumab : CD52
- all B cell cancerous cells
Why do therapeutic antibodies not work all the time (COVID)?
- mAb lost their effectiveness when new variants with mutated RBD epitopes arose. The new viral variant was not neutralized by the mAb treatment. This is because the antigen had mutated and altered, affecting the binding affinity of the mAb. e
Where can peptides come from?
Peptides can come from infected cells. Infected cells start to generate the proteins they need to survive, but using the endogenous route, the cells are able to take the protein, chop it into peptides and put it in an MHC molecule (class I in this case) and presented to a T cell
An aB T cell will only respond to a cell that presents ___ and ____
An aB T cell will only respond to a cell that presents peptide in MHC
The T cell is MHC restricted
What are the MHC molecule classes
MHC I and MHC II
What are the different types of T cells that respond to the different MHC molecules?
- CD8 T cells respond to MHC I. Any nucleated cell can present MHC class I
- CD4 T cells respond to MHC II. Only B cells, macrophages and dendritic cells can respond
- CD4 binds to the B domain of MHC II molecule
Antigen processing
Is the first step. It is generating peptides from protein-containing molecules in a cell and loading the peptides onto new MHC molecules.
Antigen presentation
This is the second step. Sending loaded MHC molecules to the surface and displaying peptide antigens in MHC molecules on the surface
What are the two sources of antigens when making peptides?
Exogenous and Endogenous
Exogenous
- Source of the antigen is from outside the cell
- These are loaded onto MHC II
Endogenous
- Source of the antigen is within the cytosol of the cell
- These are loaded onto MHC I
Why should a cell present a peptide to a T cell?
- To show killer T cells what antigens are in a host cell
- To get instructions (help) from a T cell
- To start a new T cell response
To show killer T cells what antigens are in a host
- CD8 T cells are killer cells
- Showing antigens from pathogens within the cytosol (such as viruses) allows killer T cells to eliminate the infected cell (also works for showing tumor antigens to rid tumor cells)
- Using MHC I. Any nucleated cell does this
To get instructions (help) from a T cell
- By showing pathogen antigens that are in phagosomes/endosomes
- Using MHC II, infected macrophages can show the helper T cell its contents and it can get instructions to activate and kill what is inside
- B cells get their instructions from cognate Tfh cells to activate using MHC II
To start a new T cell response
- By showing threats to naive T cells in lymph nodes. Dendritic cells use MHC I AND MHC II to activate naive CD8+ and CD4+ T cells
*Basically, the dendritic cells are at the site of infection and they are phagocytes but they don’t stick around once they get activated from a MAMP such as LPS. Once it gets activated, it starts to pick up some E.coli, process it, and it goes to an open ended lymphatic vessel that’s draining all the fluid in the inflamed area. It will then present to the T cells.
A Naive T Cell
Has never encountered its antigen in the periphery. When it is activated by a DC in secondary lymphoid tissue, it becomes an antigen-activated effector T cell*
Dendritic Cell (cDC)
professional antigen-presenting cell (also a phagocyte) that is best at activating naive T cells because it has multiple ways to process and present antigens and provide co-stimulation
MHC I Structure
- Has 3 subunits for its alpha chain (which holds the peptide)
- Has B2m chain which is an invariant (never changing) chain that is required to hold an MHC I alpha chain in the correct conformation to bind peptides. It is always a part of a conventional MHC I molecule on the surface of a cell
MHC II Structure
- Alpha chain
- Beta chain
What do both MHC structures have in common?
- Each MHC ‘class’ has an alpha and beta chain and similar folded structure since they have a similar function - presenting a short peptide to T cells
- They have folded domains that form a peptide binding cleft.
- They use hydrogen and ionic interactions
What is actually causing the binding between the peptide and the MHC molecule?
- The side chains of amino acids of peptide interact with side chains of amino acids of the sides and floor of the MHC cleft. This holds the peptide in the cleft =
How many long of a peptide can MHC I hold
- It can hold a peptide that is 8-9 amino acids in length
Anchor residues for MHC I
- Only the amino acids at the beginning and end of the peptide can interact with the MHC molecule’s tyrosine side chain. These are called anchor residues. This allows for the rest of the peptide to bind to the TCR.
What are the side chains of the MHC I molecules that bind to the peptide?
Tyrosine side chains are used by ALL MHC I to bind one end of the peptide
How many long of a peptide can MHC II hold?
22-25 amino acids long. Sometimes it can spill over the MHC cleft
What type of bonding is in both MHC I and MHC II?
Hydrogen bonds and ionic interactions (non-covalent interactions)
How is the binding of the peptide and MHC different for MHC II compared to MHC I?
MHC II doesn’t just have non-covalent interactions at the beginning and the end, it has it all throughout its peptide unlike MHC I which only has it at the end points. There are still some binding sites for the peptide to bind to the TCR once its already bond to MHC
Exogenous compartment
Endosomes and other vesicles, Golgi, ER in cells which come into contact with exogenous material
Endogenous compartment
Nucleus, autophagosome and cytosol which never come in contact with the extracellular environment
What is the housekeeping that a proteasome does?
- Cells are always making proteins and they have a housekeeping system where they mark proteins that are used up or not folded properly and destroy them with their own proteasomes. They take in proteins at one end and shoot out peptides at the other
- If you have proteins, they will all eventually be tagged with ubiquitin and it will be tagged onto proteins that are misfolded or old proteins. ANd when you add more than one ubiquitin, you start polyubiquitination
- Proteasomes have one end that binds to poly ubiquitin protein (it binds better the more ubiquitin ) and in the core are proteases that are chopping up
Ubiquitin
Small, ubiquitous protein that is attached to proteins in the cytosol. One purpose is to tag things for removal
How does the immune system co-op the proteasome
- The immune system takes some of the peptides from the housekeeping* proteasomes to put onto MHC I molecules
- So T cells can survey the proteins within a cell all the time even when the cell is not infected
- This is a constitutive proteasome (always working)
How does a proteasome change when a threat is detected?
- When threats are detected, the proteasome is changed to one that cleaves peptides differently.
1. Induced by treats that induce IFN-y for instance (from NK and T cells for example)
2.Improved cap and different proteases
3. Produces new peptides that are more suited for binding MHC I (cleaved after hydrophobic and basic residues which makes peptides with the correct anchor residues for MHC I molecules)
4. Proteins are cut at amino acids that make for “better” peptides… more suited for binding TAP
Where do newly made MHC’s wait ?
In the ER
How does B2m bind with the alpha subunits to make the MHC I molecule?
- Originally, the alpha subunits are held by a chaperone protein called calnexin that holds it open so the B2m can bind and snap it in place. B2m will hold the cleft open in just the right conformation
What complex is formed once the MHC I molecule is formed?
Once the calnexin leaves, you have a complex that’s formed by binding to the MHC. Calreticulin, ERp57, Tapasin, and TAP
What binds to TAP?
Tapasin
TAP Complex
Transports only peptides that are suited for MHC I
- 8-16 amino acids long
- has hydrophobic or basic residue at amino terminus
ERAPS
Trim the transported peptides to 8 or 9 amino acids to fit within the peptide-binding cleft of MHC I
How does a peptide actually get added to an MHC molecule?
- Inside the cytosol, a proteasome will be cleaving proteins into peptides (if it is an immunoproteasome, it will be better at this by cleaving at the hydrophobic/basic ends and making sure the protein is shorter). Peptides that are the right size (8-16 amino acids long) for TAP, will be bound and translocated into the endoplasmic reticulum
- Here, they will be met by an ERAP which will cut the amino acid sequence in to 8-9 amino acids.
- The peptides can then try to fit in the MHC cleft. Already bound to the alpha 2-1 helix of the MHC is a tapasin. If the peptide has a greater binding affinity to the alpha 2-1, then it will outcompete the tapasin, releasing it and the rest of the chapterons.
Is an ERAAP the same as ERp57
no
TAP complex
Several TAP1 and TAP2 (together TAP 1 and 2 actually make the TAP molecule) molecules that span the membrane of the ER. It is a selective gate that actively transports select peptides
What is the role of the chaperone proteins
- Only peptides that bind strongly to MHC I are loaded (tapasin)
- empty MHC I molecules never go to the cell surface
What does the Tapasin-ERp57-calreticulin complex do specifically?
- Anchors empty MHC I molecule near the tap
- Holds open the peptide binding cleft
- Competes with proteins in the binding cleft
Chaperone
A protein that assists with the correct folding, localization, or binding of other molecules. Some chaperonins compete with peptides for binding to an MHC molecule
What happens to MHC molecules that lose peptide quickly?
They unfold and won’t bind to TCR
Which cells are professional APCs?
- Macrophages, B Cells, and Dendritic Cells (cDCs and Langerhan)
How are peptides for MHC II generated?
- Antigen is taken up from the extracellular space into endocytic vesicles
- In early endosomes of neutral pH, endosomal proteases* are inactive (it would have fused with other endosomes that had inactive proteases and a H+ pump)
- The endosome will eventually fuse with a lysosome or other endosomes with pumps that will acidify the vesicle and activate the proteases
- Acidification of the vesicle activates the proteases and cuts the protein into fragments (peptides)
- Eventually, the vesicle containing the peptide will fuse with a vesicle for MHC class II.
How is MHC II loaded with peptide?
- The invariant chain (li) is a chaperone for MHC II. It stabilizes empty MHC II in the ER and blocks the cleft from loading peptides in the ER
- From the ER, the MHC II molecule and li will bud off to make a vesicle in the cytosol
- When MHC II and invariant chain vesicle fuses with an acidic vesicle, it will degrade the LIP10 portion of the li and leave the CLIP in the binding site
- This vesicle will then fuse with the acidic vesicle that now contains the peptide. However, at this point in time, the CLIP is still bound to the cleft, preventing peptides from just binding
- HLA-DM is a chaperone protein for MHC II vesicles. It competes with peptides to bind (like tapasin did for MHC I). Retains unloaded MHC II in the MIIC compartment until a peptide can bind with high affinity and knock off HLA-DM
- When the peptide binds, it’ll reclose the MHC II molecule tightly
MIIC Compartment
Acidified endosome containing degraded antigens and the chaperone, HLA-DM
Where do empty MHC II molecules go?
- They are escorted by chaperones to a special vesicular compartment for peptide loading (once they leave the ER)
- Only MHC II with peptide is released from the chaperones to go to the cell surface
Types of Dendritic Cells
- Conventional dendritic cells (cDCs) in tissues or in lymph nodes and other secondary lymphoid tissues. Subsets: cDC1, cDC2, mucosal CD103+DC (ACTIVATE T CELL)
- Langerhans cells immature dendritic cells that are sentinel cells in the epidermis that collect antigen and take it to nearest lymph node (ACTIVATE T CELL)
- Monocyte-derived DCs arise in inflamed sites from activated macrophages and take antigen to lymph nodes
- Plasmacytoid dendritic cells don’t activate T cells but they secrete Type I IFNs
- Stromal follicular DC don’t activate T cells they activate B cells
What does it mean for a cell to be dendritic?
Having a branched cell morphology (like branches of a tree). Different types of dendritic cells have very different functions
How do immature DCs get activated?
- They recognize MAMPS with PRRs such as TLRs, NOD and NLRs, and RLRs)
- PRR signaling activates the cell to migrate and change activities
What changes are there in a mature DC versus an immature DC?
- DC change chemokine receptors to CCR7 to migrate to T cell areas of the lymph node that are rich with chemokines CCL19 and CCL21
- It also enhances processing of pathogen-derived antigens
- once they recognize these chemokines, they know they are in the lymph node
What does a mature DC express to activate a T cell?
- Mature DCs are processing antigens as they migrate towards the lymph nodes
- They express b7, MHC, and ICAM 1
- They present antigen and secrete cytokines to activate T cells
DIfferent stages of DC activation
- Immature: sentinel at barrier, function is to take up antigens and microbes and sense MAMPS
- Activating: a migratory cell, function is to migrate and start to process antigen
- Fully mature: a cDC, function is to present an antigen with MHC molecule, express costimulatory and fate-specifying cytokines
Mature conventional DC present antigens by many routes
- Endocytosis and pinocytosis of exogenous antigens
- Viral infection of a DC allows the unusual endogenous antigen processing pathway
- Cross-presentation, cDC1 can transport endocytosed antigen across cytosol to present it on the endogenous MHC I path
- Dying cells are phagocytosed by CDC1 and their antigens are cross-presented on MHC I
- Other DC (like activated Langerhans cells) arrive at the LN and transfer antigen to cDC1 that cross-present antigens on MHC I
What are the two different ways of presenting an antigen?
- Direct
- Indirect
Necrosis
A very dangerous form of cell death that activates cross-presentation because it releases LOTS of DAMPS
What if a virus cannot infect a dendritic cell? How would CD8+ killer T cells ever be activated? (like herpes or influenza)
Dendritic cells can still engulf the antigens, process them, and switch them to the endogenous route so that they can be presented on MHC I molecules to CD8 T cells
What if dying cells can’t migrate to the lymph node
Langerhan cels can take the dying cell, bring it to a cDC and it can be processed to it can be expressed to T cells
Which cells are the only ones that can truly alert both CD4 and CD8 T cells?
cDC1
When does cross-presentation occur?
- cDC1 will cross-present antigen ONLY when they are activated by IFNs that announces an intracellular threat
- Type I and type II IFNs induces the genes that allow cross-presentation (and use of immunoproteasomes)
How many CDRs are on one antigen binding site
6 for both B and T cells
Locus
A cluster of genes
What is the Classical MHC I chain encoded by?
- HLA-A
- HLA-B
- HLA-C
- the gene only codes teh alpha portion of the MHC molecule, the B2m gene is encoded elsewhere
What is the Classical MHC II chain encoded by?
- HLA-DPA, DPB
- HLA-DQA, DQB
- HLA-DRA, DRB
Major Histocompatibility Complex (MHC)
A set of genes that are largely involved in antigen processing and presentation. And other immune function
Classical MHC genes
Encode chains of MHC molecules that present peptides to aB T cells
There are many immune genes in the MHC complex
- Inactive duplication (pseudogenes, for instance)
- Many functions besides holding peptide
- Some genes encode proteins with the same function… DRB1 and DRB3 encode two functional Beta chains that could be used in an MHC II molecule
Some MHC-associted genes are on other chromosomes like the gene encoding B2m
What is the Mouse nomeclature for their MHC complex
H-2
MHC Class I: H-2K, H-2D, H-2L (missing in some strains)
MHC Class II: H-2A (I-A), H-2E (I-E) `
How many genes encode all MHC that present antigen to aB T cells?
Only 10 genes
What are the four ways that MHC genes become so diverse
Polygeny
Polymorphism
Codominant expression
Promiscuous specificity
Polygeny
Use multiple genes for multiple proteins with the same function
Polymorphism
Make each gene variable between individuals so there are MANY versions (alleles) to present MANY different peptides in a population
Co-dominant expression
Inherit and express both chromosomal copes of MHC genes to “double-up:
Promiscuous specificity
Design each MHC molecule so it can bind a set of related peptides that have similar anchor residues
How many different peptides can a person present?
Typically, a person expresses 12-14 different classical MHC I and MHC II molecules. Each bind about 10,000 different peptides
(12 to 14) x 10,000 = 120,000 to 140,000 different peptides presented in a person
Do we all express the same 120,000 to 140,000 peptides>
People don’t present the same peptides
- Offspring inherit different chromosomes that have different alleles of MHC molecules that present different sets of peptides. So, a population is diverse
- The 120,000 to 140,000 presented peptides are somewhat different between individuals in a population
Haplotypes
MHC locus on one chromosome (one from mom and one from dad)
Different MHC gene alleles on different chromosomes
- Children inherit different combinations of chromosomes
- EACH CHILD SHOULD HAVE DIFFERENT HAPLOTYPES
- They usually end up heterozygous for their MHC haplotypes
- And they will express both copies (co-dominant expression)
If the genes are the same what does the allele gene do? If they are different what do they do?
If they are different (ex, DP 2-9), then the allele will make two different allotype proteins. If they are the same (ex. DP 10,10), they will not
Example of polygeny in MHC
HLA-A, HLA-B, and HLA-C are proteins that have the same function but different genes
Example of polymorphism
There are 2980 allotypes of HLA-A. You could have HLA-A23 or HLA-A56
What are HLA-A, HLA-B, and HLA-C besides a polygeny
Isotypes of MHC I
What do the non-classical HLA molecules do? (MHC I)
HLA-E, F, and G are used to bind NK receptors
What do the non-classical HLA molecules do? (MHC-II)
HLA-DM and HLA-DO are used for loading peptides into MHC II molecules
So, 2 chromosomal haplotypes means
6 or 8 different MHC II molecules
+ 6 different MHC I molecules
is about 12 to 14 different MHC molecules on a professional antigen-presenting cell
Where do allotypes vary?
In the peptide-binding cleft
Closer look at promiscuous specificity
- Peptides are bound at certain anchor residues… such as in this MHC I cleft
- So, any peptide of the correct length with the same anchor residues or ones with similar side chains binds (this is known as the same sequence motif)
TCRs and MHC bind peptides at different residues
- Using TCR CDR 3 loops
- Us
Does everyone have the same B2m chain
Yes
Inbred mice
- All mice of an inbred strain have 2 identical copies of each chromosome… so, they have 1 haplotype (2 identical chromosomal complements of MHC genes)
The set of genes that a particular strain of mouse has is called it _____ type
H-2
ex. the H-2 type of BALB/c mice is called H-2^d while the H-2 type of C57BL/6 is H-2^b
the point of this is to show that they are identical to each other, but the two different strains of mice have different genes
Therefore, H-2K^d of BALB/c mice will hold different peptides and bind different TCRs of H-2kB of C57BL/6 mice
How are the T cells of inbred mice MHC restricted?
The T cells of an H-2k mouse are restricted to binding H-2k MHC molecules
- Mst T cells from an H-2k mouse will not bind HC of an H-2b mouse, even if it is presenting the peptide that th eT cell is specific for
- This is known as MHC-restriction (aka self-MHC restriction)
** In vitro experiments must match H-2 tyoes for T cells to lyse target cell lines, for instance
MHC I Summary (key points)
- Expressed by all nucleated cells
- Recognized by CD8+ CYTOTOXIC T cells
- Source of peptide = cytosol of cell (endogenous)
- Cleft closed at both ends (can close completely
- Peptides anchored at both ends - easy to preidct by hydrophobic residues
MHC II Summary (key points
- Expressed by ‘professionals’ (DC, Mac, B)
- Recognized by CD4+ HELPER T cells
- Source of peptide = endosomal vesicles (exogenous)
- Cleft open at both ends , longer peptide (>13 amino acids)
- Peptide anchored along length of groove, difficult to predict peptides
What are the solutions for creating antigen receptors that are very diverse?
- Use genes with many interchangeable segments that encode the anitgen-binding part of the receptor
- Randomly combine the gene segments to encode the variable antigen-binding region
- Introduce random mutations when joining the segments for additional diversity
Germline DNA
Inherited DNA that has not been rearranged by somatic recombination
When do germline genes get activated?
The germline genes are very long which makes them inactive until they are recombined
What are the light chains?
Kappa and Lambda and this is only found in a B cell
Is there more than one type of heavy chain in a B cell?
No
What segment does the Heavy chain have that the light chains don’t?
The Diversity segment
Combinatorial diversity
Randomly combine gene segments that encode antigen-binding site
ex. How many different combinations of segments are possible for a human k light chain
(38V) (5J) is 190 different combinations encoding k chain
Random pairing
Randomly pairing is randomly pairing a heavy and light chain (remember there are two light chains)
ex. (190K)(6348H) + (165 lambda)(6348H)
*random pairing is the only one with addition but she won’t ask you about random pairing
Somatic recombination in B Cells
Only immature B cells (and T cells) express the correct combination of transcription factors and enzymes to rearrange immunoglobulin genes and put B cell receptors on their surface. As plasma cells (& plasmablasts) they secrete antibodies by differentially splicing the primary RNA
What are the functions of a Pre-B cell?
- Rearrange Ig genes (somatic recombination) and express a working antigen receptor (BCR)
Somatic Recombination
DNA recombination that takes place in somatic cells. In immature B and T cells, it occurs between V D and J gene segments. It generates a complete exon that encodes the variable region of the BCR, antibody, or TCR chain
Recombination Signal Sequences (RSSs)
Conserved motifs that bind a recombinase complex. There are 2 types (one with a 23 nucleotide spacer) and (one with a 12-nucleotide spacer). The arrangement of RSSs ensures the correct type of segments are boudn and recombinds by RAGs
RSS orient the RAG complex on certain segments
- A 12-nucleotide RSS is brought alongside a 23 nucleotide RSS by a recombinase enzyme complex (RAG1/RAG2) (the RAG 1’s bind together to bring them together)
- Only RSS with different spacers will orient RAG1 correctly to form a loop of intervening genes to be cut out (excised)
*Draw this
RAG 1 and RAG 2
Genes are only expressed in developing B cells and T cells. They encode recombinases that bind a different spacer lengths (12 nucleotide and 23 nucleotide). The RAG complex cleaves genomic DNA in a cell which permanently removes the intervening stretch of the antigen receptor gene
12/23 Rule
Two RAG-1 molecules will only bind and cut out the intervening DNA when they are binding RSSs that have different spacer lengths (12-nucleotide and 23-nucleotide). The arrangements of RSSs that flank segments ensures that 2 of the same segments are never joined (like two V segments)
Explain how the coding joint between 2 joining segments is mutated and repaired when the segments join
- The RAG enzymes cut at the heptamers and open hairpins
- When the enzymes are open, they are palindromic P-nucleotides (ex. TCGA’s complement is AGCT and if you say that backwards it is TCGA)
- Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly (can add up to 6!). These nucleotides are called N nucleotides
- Common DNA repair enzymes clear mismatches and fill gaps
Signal Joint
The loop of DNA that is cut out by the RAG complex is lost from the pre-B cell as it devided into many daughter cells. All the daughter cells lack this loop of DNA, too meaning they express the exact same BCR.
Terminal deoxynucleotidyl Transferase
Enzyme that inserts nucleotides randomly (Non-coded, N-nucleotides) into gaps between V-D and D-J segments of heavy chains and all T cell receptors before they are repaired. The enzyme is active in pre-B and pre-T cells.
Junctional diversity
variation in the Ig and TCR chains created during the joining of variable gene segments by addition or removal of nucleotides at junctions between v(D)j segments. Occurs in heavy chains and all TCR chains
Which chains does junctional diversity occur in?
This always happens in the heavy chains and occasionally in light chains
If she says up to 6…
4^1 + 4^2 + 4^3 etc.
Total antigen-binding diversity
Encompasses combinatorial and junctional diversity. It is the number of possible gene sequences that encode TCR or immunoglobulins (antibodies and BCR)
Expressing the C region segment
- Only happens on heavy chain of Ig (because that encodes the isotypes)
- IgM and IgD are BOTH expressed as BCR forms on a B cell at first
Immunoglobulin gene constant gene segments
Encode non-variable constant portions of heavy and light chains. The C segments of the heavy chain locus encode the isotype (function) of the antigen receptor
Downstream of heavy chain rearranged variable regions…
Are 9 constant (C) gene segments
*there are 4 gammas for the 4 sub-isotypes of IgG
*there are two alphas for the dimeric and mono IgA
- Mu and delta are the first segments to be transcribed
How does the primary RNA impact the C region?
The primary RNA transcript is spliced in two places
- If it ends with Cmu - mRNA encodes IgM
- If it ends with Cdelta - mRNA encodes IgD
- C segments are selected at the RNA step and don’t recombine
Alternative RNA Splicing
To create mRNA encoding different immunoglobulin isotypes
- First, rearranged DNA is transcribed into a primary RNA transcript
- Then, the primary RNA transcript is spliced to yield mRNAs ending with Cmu or Cdelta segments. This will encode IgM or IgD B cell receptors
- Both are simultaneously expressed on the same B cell
Does splicing occur before or after rearrangment?
After
Primary RNA transcript
form of RNA with introns that is spliced to generate different mRNAs
mRNA
form of RNA that is translated into proteins
Splicing RNA _____ change antigen-binding region. It changes the _____ _____ to give the antigen receptor different functions, like ___ a ____ to the B cell
Splicing RNA does not change antigen-binding region. It changes the constant region to give the antigen receptor different functions, like transmitting a signal to the B cell
There are two classes of T-cell receptors. Name them
- Alpha-Beta T-cell receptor (these are always paired together)
- Gamma-delta T-cell receptor (these are always paired together)
What is special about the delta chain on a T cell receptor?
- It is found entirely within the alpha locus betwen the V and J segments
- The gamma chain is on a completely separate chromosome
What segments does the alpha chain in a TCR have?
The V and J
What segments does the beta chain in a TCR have?
The V, D, and J
What segments does the gamma chain have?
The V and J
What segments does the delta chain have?
The V,D, and J (encoded in the alpha locus)
Somatic Recombination in T cells
Only pre-T cells make the correct combination of transcription factors and enzymes to rearrange TCR genes and put T cell receptors on their surface
T cells NEVER secrete the TCR not improve the TCR which are big differences from B cells
What are the functions of pre-T cells?
Rearrange TCR genes (somatic recombination) and express a working antigen receptor (TCR)
Does junctional diversity happen in TCR too?
Yes, it is the same process with the same RAG, TdT, and common DNA repair enzymes (and 1-6 nucleotides are still added!)
What does junctional diversity create (specifically in T cells)?
- All antigen receptors have hypervariable regions that bind to epitopes. They are called CDRs
- CDR1 and CDR2 are encoded in the V gene segment
- CDR3 is encoded by the junction between gene segments… the one that has nucletotides randomly inserted and deleted when the segments are joined
How many loops does junctional diversity create?
One loop that binds antigen
How many loops bind the antigen in a TCR?
The 2 middle CDR loops bind the antigen (these are the CDR3 loops), the other 4 bind the MHC molecule (the CDR1 and CDR2)
Is the junctional diversity creating a CDR loop limited to only T cells?
No, BCR also do this. Both the light chain and the heavy chain will receive a loop if they had junctional diversity
