Antigen presentation and T-cell signalling Flashcards
LO
- Explain why the Major Histocompatibility Complex is important and their role in Antigen Presentation
- Describe the molecular basis by which T cells recognise and respond to antigen
- Identify key features of the Antigen Processing pathways
- Describe how dendritic cells are important in initiating T cell responses
Topics covered in antigen presentation and T cell signalling lectures
Why do we need antigen presentation?
How do T cell recognise antigens?
How do T cells recognise the peptide MHC complex?
- Where do MHC molecules come from?
MHC class I antigen processing pathway
MHC class II antigen processing pathway
Role of dendritic cells (DC) in initiating a T cell response
How do T cells activate the B and CD8 cells?
via cytokines
What do T cells undergo in the thymocyte?
They undergo positive and negative selection in order to become mature T cells
What types of cells are the T cells when they leave the thymus, why are they described in this way?
How do they change when an infection is present?
When the T cell leaves the thymus they are mature and naïve, because they haven’t been exposed to an antigen yet
Once exposed to an antigen they become mature and primed
Then they can start to proliferate and and move to the area of infection so that they can start to help to fight towards it
What is lymphocyte recirculation?
Lymphocyte recirculation refers to the continuous transport of large numbers of naïve lymphocytes between the blood and lymphatic systems.
After naïve lymphocytes are produced in the thymus or bone marrow, they enter the blood, where they spend about 30 min, before migrating to the lymphoid and nonlymphoid organs.
This process is key for the immune response
Tell me the stages to T cell priming and maturing?
How do T cells recognise antigens?
T cells recognise antigenic fragments bound to MHC molecules
MHCI interact with CD8
MHC are expressed on all cells
MHC1 Are not exposed on RBC or neuronal cells, but they are on other cells
MHCII are only expressed on professional APC
MHCII present to CD4
Explain the similarities and differences between the MHC class I and class II molecules tertiary structure
Similarities:
- Both types of MHC molecule that are encoded by gene clusters of MHC
- Both MHC I and II are surface antigens that are expressed on the cell membrane
- Both present antigens to T cells
- Both are involved in the development of immune responses against foreign antigens
- Both are responsible for graft rejection during various organ and tissue transplantation
Differences:
- MHCI found on surface of all nucleated cells Vs MHCII found professional APC
- MHC I composed of 3 alpha domains and 1 beta domains Vs MHCII composed of 2 alpha and 2 beta domains
- MHCI alpha domains encoded by locus of chromosome 6 Vs MHCII endoed on the chromosome 6
- MHCI present endogenous antigens originated from cytoplasm Vs MHCII present exogenous antigens originated extracelluarly from foreign bodies such as pathogens
- MHCI alpha 1 and alpha 2 are involved in presentation of antigens vs Alpha 1 and beta 2 domains which are involved in the antigen presentation
- MHCI present antigens to CD8 cells vs MHCII present antigens to CD4 cells
What is the structure of the MHCI molecule?
Heavy chain comprised of alpha1, alpha2 and alpha3 domains
Heavy chain tethered to membrane via cytoplasmic tail
The beta2-microglobulin does not have a transmembrane region but it does stabilised the peptide binding groove
What is the structure of the MHCII molecule?
Heavy chain is comprised of alpha1, alpha2, beta1 and beta2 chains
Because these 4 make up the peptide binding group it means that the conformational binding is different to that as found in class I
alpha 2 and beta2 have the transmembrane cytoplasmic tails
TCR and MHC class I molecule
Tell me about the peptide binding groove in the class I and class II MHCs
In MHCII the Peptide binding groove can bind peptides longer than the MHC molecules. Therefore bind a more diverse group of peptides which is important for immune response
Each chain has two domains and altogether form a four-domain heterodimer similar to the MHC-I molecule. α1 and β1 domains form the peptide-binding cleft resulting in a groove which is open at the ends, which is different from the MHC-I groove in which the extremes of the peptide are buried at the ends.
What determines the type and size of a peptide that binds into the groove of a MHC molecule?
The polymorphic residues that line the the peptide-binding groove determine the peptide-binding properties of the different MHC molecules
Tell me what involved in peptide antigen binding
MHC can bind a wide range of peptides
Anchor residues for H-bonds to bind peptide into groove
Peptides can adopt different conformations and ‘bulge’ out of groove- allowing TCR to sense peptide
Bulge is dependent on AA sequence and the length of peptide
TCR cannot distinguish what AA are but can distinguish the type from bulge formed?
What type of sequencing can be done to identify allele-specific anchor residues?
Explain this process
Pooled sequencing e.g.., HLA-A2
Pooled sequencing of eluted peptides from HLA molecules expressed at the cell surface
Identified important features of anchor residues using mass spectrometry
image shows has preference for hydrophobic AA at p2 and p9
Take HLA-A2 and elute proteins from then, then use mass spec, find is residues are preferentially bound to this molecule or another HLA molecule. above shows they prefer hydrophobic residues
What are MHC molecules important for?
important in establishing immune responses to infection, autoimmunity and cancer
What can MHC molecules determine?
Your ability to respond to infections e.g., HIV
Tell me about MHC/ HLAs involvement and role with tissue grafts
An important cluster of genes that control tissue graft survival in transplantation (key to have HLA matching for transplantation in order to avoid rejection)
MHC referred to as human leukocyte antigen (HLA) in humans
Antigen presentation and the type of MHC you have is important in autoimmune diseases?
What three terms can be used to describe the characteristics of MHC diversity?
polygenic
polymorphic
co-dominant
Explain the three characteristics of MHC diversity
Characteristics of MHC diversity
MHC are polygenic
- Multiple genes with the same function but slightly different structures: broad range of peptide binding specificities
MHC are polymorphic
- Multiple forms of each gene exist within the population (termed alleles)
MHC are co-dominantly expressed
- Set of linked alleles (haplotypes) inherited from parents
Why is the MHC described as being a 6-digit barcode?
Its comprised of 6 genetic loci
Tell me about the MHC locus
B, C, A, K, D, L are MHC class I
DP, DQ, DR, A, E are MHC class II
Human HLA and mouse H2?
In the MHC 6-digit barcode, it is comprised of 6 genetic loci. Each locus is co-dominantly inherited and contains/ expresses what?
Each locus Is co-dominantly inherited…
- Two of each of the 6 HLA are expressed
- MHC/ HLA haplotype (a set of genetic determinants located on a single chromosome)
Tell me the nomenclature system of HLA
All alleles start with “HLA”, signifying they are part of the human MHC genes.
The next portion (HLA-A or HLA-B) identifies which gene the allele is a modification of.
The first two numbers (HLA-A*02) signifies what antigen type that particular allele is, which typically signifies the serological antigen present.
What do the MHC Polymorphisms encode?
The differences in the peptide-binding groove
What do the different MHC groove binding polymorphisms permit?
Binding of the different peptide repertoires
Summary of MHCI and MHCII
What does T cell activation also require co-receptor interaction with?
MHC
How does the TCR/MHC interaction communicate inside the cell for activation?
TCR complex
What does the TCR associate with that has conserved multi subunit signalling apparatus?
The CD3 complex
Describe the stages to the T cell signalling cascade
- T Cell Receptor (TCR) activation promotes a number of signaling cascades that ultimately determine cell fate through regulating cytokine production, cell survival, proliferation, and differentiation.
- An early event in TCR activation is phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein tyrosine kinase (Lck).
- The CD45 receptor tyrosine phosphatase modulates the phosphorylation and activation of Lck and other Src family tyrosine kinases.
- Zeta-chain associated protein kinase (Zap-70) is recruited to the TCR/CD3 complex where it becomes activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins.
- Phosphorylation of SLP-76 by Zap-70 promotes recruitment of Vav (a guanine nucleotide exchange factor), the adaptor proteins NCK and GADS, and an inducible T cell kinase (Itk).
- Phosphorylation of phospholipase C γ1 (PLCγ1) by Itk results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3).
- DAG activates PKCθ and the MAPK/Erk pathways, both promoting transcription factor NF-κB activation.
- IP3 triggers the release of Ca2+ from the ER, which promotes entry of extracellular Ca2+ into cells through calcium release-activated Ca2+ (CRAC) channels. Calcium-bound calmodulin (Ca2+/CaM) activates the phosphatase calcineurin, which promotes IL-2 gene transcription through the transcription factor NFAT.
- Feedback regulation at several points within these pathways allows for different outcomes, depending on the cell type and environment.
- The incorporation of signals from additional cell surface receptors (such as CD28 or LFA-1) further regulates cellular response.
What are the steps to the PLCgamma pathway?
- Phospholipases, in particular PLC-γ, are important effector enzymes for membrane-bound receptors
- PLC-γ contains SH2 domains that function as interacting domains for tyrosine phosphorylated RTKs.
- This allows PLC-γ to be intimately associated with the signal transduction complexes of the membrane as well as membrane phospholipids that are its substrates.
- Activation of PLC-γ leads to the hydrolysis of membrane PIP2 resulting in an increase in intracellular DAG and IP3 as second messengers.
- The released IP3 interacts with intracellular membrane receptors leading to an increased release of stored calcium ions (discussed below).
- The primary signaling molecule affected by the activation of phospholipases is PKC, which is maximally active in the presence of calcium ion and DAG.
- Although the principal mediators of PKC activity are receptors coupled to activation of PLC-γ, phospholipases D and A2 (PLD and PLA2) may also be involved in the sustained activation of PKC through their hydrolysis of membrane phosphatidylcholine (PC)
- PLD action on PC leads to the release of phosphatidic acid, which in turn is converted to DAG by a specific phosphatidic acid phosphomonoesterase.
- PLA2 hydrolyzes PC to yield free fatty acids and lysoPC both of which have been shown to potentiate the DAG-mediated activation of PKC
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Tell me the steps to the GEF associated pathway
Ras-GDP is inactive
GDP exchange factor facilitates exchange and makes Ras-GTP which is active
This acts on Raf –> MEK –> MAP kinase
Which goes onto act on FOS which forms a complex with Jun and activates transcription activation of several genes
Activation and proliferation of CD4 and CD8 T cells
Where do MHC molecules come from?
Antigen processing and presentation pathways
Left is the exogenous MHCII pathway which presents to CD4 T cells
Right shows endogenous MHC I pathway which presents CD8 T cells
Peptides for MHCII are derived from outside of the cell
They are digested from outside of the cell, presented on MHCII and then loaded onto cell surface
Tell me about MHCI antigen processing pathway (endogenous peptide antigens)
Shows overview of pathway itself
Antigenic peptides start as proteins in cytoplasm of cell
Proteins are formed and folded where they are loaded and transported to the outside of the cell
TAP transport is associated with antigen processing?
Good C terminus for binding and extended N terminus in cytoplasm before ERAP comes along as a ‘pacman’ and trims the N-terminal AA. This generate the repertoire peptides that are seen on the cell surface
ERAP is highly polymorphic and highly variable
Polymorphisms change the function of ERAP activity
Peptides are loaded in peptide loading complex
TAP will exchange low affinity peptides for high affinity peptides in peptide binding groove
Peptides have to bind with high affinity to MHC in order to be dissociated and be expressed on cell surface
MHC have to be on cell surface long enough for T cells to respond
Tell me about the origin of peptides for MHCI
Not limited to MHC I pathway, but this naturally occurs in cell and keeps a natural homeostatic environment
Proteasome can chop proteins into smaller peptide fragments which is important for MHCI processing
Target protein is threaded into proteasome
Ubiquitin is released
Tell me about the proteasomes function, activites and specificity
Function: Degradation of unfolded or Ubiquitin-tagged cellular proteins
Three activities: cleaving after basic, acidic, and hydrophobic/aromatic residues
Specificity: In IFN-g-stimulated cells, change in activity favours the generation of peptides with C-terminal ‘anchor’ residues for MHCI
How do peptides enter the endoplasmic reticulum?
via the TAP transporter
Tell me about Tumour antigens
Tell me the overall outline to MHC class II antigen processing pathway (exogenous)
Antigens derived from outside of the cell
Antigen is engulfed by the membrane and is in a vesicle within the cell known as an endosome or lysosome
MHCII are formed in the ER
Where are cathepsins found and what are they?
They are found in vesicles with low ph
Cathepsins are a type of protease
Antigen –> APC
Antigen processing and presentation in T cell activation
Whats the role of dendritic cells (DC) in initiating a T cell response
How do T cells encounter peptide antigen?
Dendritic cells are professional APC which present MHCI and MHCII. Predominantly MHCII
Once T cells primed –> differentiation –> go to site of infection
Tell me about the distribution of MHC molecules
MHC are expressed on immune cells as well as other cells in the body
The only cell that doesn’t is neuronal cells
The professional APC are the only ones that present MHCII?
Massive expression levels that DC cells have MHCI and MHCII. So they are the key cell that interacts with T cells in lymph nodes
Tell me about cross presentation
- 3rd pathway of antigen processing and presentation
- Exogenous antigens are internalised and presented on MHCI
Vacuolar pathway
Classical MHC I pathway
- Exit phagosome through unknown ‘?’ mechanism, know It happens but it is unknown
- Only really happens in DC as can internalise pathogen and present on MHC
What do pathogens detect that helps with the immune response
Dendritic cells detect PAMPS and DAMPs through several classes of surface and intracellular receptors called pattern recognition receptors (PRRs). These classes include the receptor for advanced glycation end products (RAGE), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and Toll-like receptors (TLRs).
Whats an example of a pattern recognition receptor (PRR) and tell me about it?
Toll-like receptors (TLR) are a type of PRR
- Germ-line encoded receptors
- Expressed on surface of innate cells
- Are highly specific but have a broad binding profile- recognise many different pathogens
Tell me about Toll-like receptors and DC activation
Tell me the stages to TLR signalling in order to activate dendritic cells
- The components of the TLR signalling pathway
- TLR-4 is bound to LPS binds the adaptor protein MyD88
- MyD88 binds and activates SIIK, which initiates a cascade of TRAF6 and the kinase IkappaK
- IkappaK phosphorylates IkappaB
- Phosphorylated IkappaB is degraded, releasing NFkappaB to migrate into the nucleus and activate gene transcription
NOTE: IkappaB is bound to NFkappaB
Where do activates dendritic cells migrate to?
lymph nodes
Here they interact with T cells in the paracortex and this is where the interaction of CD8 and CD4 T cells also occur
Tell me the 3 steps to T cell activation and what each of the steps help with
First step: TCR: MHC
Second step: stimulation upregulation i.e., B7. Interacts with protein or ligand expressed on CD4 or CD8 T cells such as CD28?
Third step: cytokine release from DC which aid in activation of CD4 and CD8 T cells
All three needed to activate CD8
CD8 needs extra stimulus from CD4 cells so not activated at wrong time and place
How do primed T cells know where to go after they leave the lymph node?
Molecules present on DC which are present on different areas of the body
The upregulation of molecules on T cells due to interaction with DC cells allow them to home into where they are needed
What do tissue specific cues promote?
Tissue specific cues promote the upregulation of relevant homing receptors
