Cellular adaptive immunology Flashcards
Lectures: -Week 1, day 2, lecture 4: MHC-HLA system -Week 1, day 2, lecture 5: Antigen presentation -Week 1, day 3, lecture 3: Adaptive immune response - Cellular -Week 3, day 3, lecture 3: Developmental aspects - T-cell development
How is the adaptive immune response started?
Presentation of pathogen derived peptides, in the context of an MHC-molecule, to naïve T-cells
In which two ways do pathogen derived peptides reach naïve T-cells in the lymph node?
- Active transport -> immature DC’s pick up pathogen (or pathogen fragments), mature and move through the lymphatic vessels to regional lymph nodes, where they activate T-cells, which further activate the immune response
- Passive transport -> pathogen fragments passively get transported to the lymph nodes via the lymph, where they get picked up by B-cells, which active T-cells. The T-cells then produce cytokines that active the B-cells.
What is the function of MHC-molecules?
Presentation of pathogen epitopes to the (adaptive) immune system
Which class of T-cells is activated by MHC-I?
CD8+ T-cells (cytotoxic T-cells)
Which MHC-I molecules are there?
HLA-A, HLA-B & HLA-C
In which part of the MHC gene locus are MHC-I genes encoded?
Class I region
Which class of T-cells is activated by MHC-II?
CD4+ T-cells (T-helper cells)
Which MHC-II molecules are there?
HLA-DP, HLA-DR & HLA-DQ
In which part of the MHC gene locus are MHC-II genes encoded?
Class II region
How many alleles of MHC-I are present in the MHC gene locus?
1 allele of each HLA-A, HLA-B & HLA-C
How many alleles of MHC-II are present in the MHC gene locus?
2 genes for each of the molecules (HLA-DP, -DR, -DQ): a heavy-chain/α-chain and a light chain/β-chain per chromosome
Where is the MHC gene locus located?
Short arm of chromosome 6
What is the structure of MHC-I? Where is the peptide binding cleft located?
An α-chain consisting of 3 domains, supported by a β-microglobulin
The peptide binding cleft is located between α1 & α2
What is the function of the β-microglobulin of MHC-I?
Stabilization of MHC-I on the cell surface
True or false: the β-microglobulin of MHC-I is encoded by the MHC gene locus
False, β-microglobulin is encoded on chromosome 15
How big are peptides presented by MHC-I? In what way do they lie in the peptide binding cleft?
Peptides are between 8-10 amino acids long
The peptide is fully contained within the peptide binding cleft
What is the structure of MHC-II? Where is the peptide binding cleft located?
One heavy chain = α-chain, consisting of 2 domains
One light chain = β-chain, consisting of 2 domains
The peptide binding cleft is located between the α1 and β1 domains
How big are peptides presented by MHC-II? In what way do they lie in the peptide binding cleft
Peptides are at least 13 and mostly between 13-17 amino acids long
The peptide extends beyond the peptide binding cleft
On what cells is MHC-I expressed?
All nucleated cells
On what cells is MHC-II expressed?
APC’s (DC’s, B-cells and macrophages)
Which part of MHC-II interacts with CD4 on the CD4+ T-cell?
The β-chain
Which part of MHC-I interacts with CD8 on the CD8+ T-cell?
The α-chain
HLA-genes are highly polymorphic. What effect does this have on the characteristics of the HLA-molecules?
Leads to amino acid substitutions in the peptide binding cleft, changing the binding properties of the cleft
Where is polymorphic variation in MHC-II seen?
Rarely in the α-chain, often in the β-chain
Mostly in the peptide binding region (β1-domain) of the β-chain
Where is polymorphic variation in MHC-I seen?
Mostly in the domains that make up the peptide binding cleft -> α1 & α2
What are anchor residues in the context of MHC-molecules?
Specific amino acid residues present at specific sites in the presented peptide that allow it to bind to the MHC-molecule
Which class of MHC has the most strict anchor residues?
MHC-I -> MHC-II is more flexible in its anchor residues
True or false: anchor residues on presented peptides always have to be the same amino acid
False; they have to be of the same class of amino acids, but not strictly the same amino acid
What is the downside of the less strict anchor residue binding of MHC-II when it comes to research?
It makes it more difficult to predict which peptides will bind to MHC-II
What is MHC-/HLA-restriction? What does this mean for the specificity of the TCR?
T-cells only recognize their antigen when they are presented in the context of a particular self-MHC molecule
The TCR is specific for both the antigen presented & the type of MHC bound to the peptide
Expression of HLA-genes is codominant. What does this mean?
Both maternal and paternal alleles are expressed, resulting in the presence of maternal and paternal HLA on cells
Which two mechanisms contribute to the diversity of HLA expressed in an individual?
Polymorphisms in the HLA-genes
Polygeny -> codominant expression of maternal and paternal genes
What is the clinical significance of the existence of different HLA-serotypes (e.g. HLA-B27)
Some serotypes are associated with disease, transplant rejection and drug hypersensitivity
In which ways can the HLA-type be determined? (3)
- Serology -> adding antibodies against specific HLA types to cells, adding complement and detecting lysis of cells
- Flowcytometry
- PCR to type at DNA level
Which three signals do DC’s need to give to T-cells to achieve proper activation?
- Ag-specific activation via MHCII to TCR
- Activation of specific coreceptors
- Cytokines that cause polzarization -> determines the type of Th-response
What are the steps of antigen processing? (3)
- Antigen uptake
- Antigen processing -> generation of peptides from intact protein
- Presentation on surface MHC-molecules
In which ways can a DC take up antigen? (2)
- Direct infection of the DC
- Phagocytosis of extracellular pathogens
Antigens can be processed in different compartments of the DC. In which compartment is which type of pathogen processed?
- Cytosol processes viruses
- Vesicular systems process extracellular bacteria & parasites
Where do peptides presented in MHC-I originate?
Intracellular derived peptides, including self-peptide (in general)
Where do peptides presented in MHC-II originate?
Extracellular derived peptides (in general)
The TCR has to interact with MHC at several occasions in order to produce a functioning, active T-cell. What are they?
- During T-cell development in the thymus
- Activation of naïve T-cells in the lymph nodes
- T-cell effector function in the infected tissue or lymph nodes
What is the vesicle that contains a phagocytized particle called?
Endosome
In the processing of peptides to be loaded onto MHCII, the endosome has to fuse with 2 compartments. What are they and what are their functions?
- Lysosome, allows for breakdown of proteins into peptides
- MIIC = MHC-II containing component, contains MHC-II molecules and proteases required for peptide processing
MHC in the MIIC-component is chaperoned. Which protein functions as chaperone?
The invariant chain
What are the functions of the invariant chain? (3)
- Stabilizes MHC-II
- Prevents self-antigen from being loaded into MHC-II
- Guides MHC-II to MIIC
What happens with the invariant chain when the MIIC fuses with the phagolysosome?
The invariant chain is cut into CLIP = class-II associated invariant chain peptide
What is the function of CLIP? Which protein is involved?
CLIP is be swapped for exogenous peptide by HLA-DM
By which protein is MHCI stabilized?
β-microglobulin
Where are antigens that are due to be loaded in MHCI processed?
Proteasome
Where is MHCI loaded with peptide? How do peptides reach this location?
The ER (where MHCI is produced)
Peptides are transported into the ER by TAP1/TAP2
What is cross presentation? What is its function?
Cross presentation is the presentation of extracellular antigens on MHCI by professional APC’s. This is neccesary to be able to prime CD8+ T-cells
What are the three cross presentation pathways?
- MHCI is present in the endosome/phagolysosome and is loaded with peptides there
- Export of extracellular antigen to the proteasome, via which it ends up in the cytosolic pathway and gets loaded into MHCI in the ER
- Antigen is sent to the proteasome, cleaved and then sent back to the phagosome
How does MHCI end up in the endosome/phagolysosome in the cross presentation pathway?
Likely because MHCI is associated with invariant chains that guide it to the MIIC, which fuses with the endsome
Why is it advantageous for macrophages to activate CD4+ T-cells when they have phagocytized pathogens?
The Th-cells will assist in killing the intravesicular pathogen
Why is it advantageous for B-cells to present antigen to Th-cells and activate them?
The activated T-cell will activate the B-cell and assist it in antibody production (Th2-cells)
What is autophagy? What is its role?
Delivery of cytosolic products to the MIIC, where they are loaded into MHCII. This ensures that the cell displays self-peptides to CD4+ T-cells
Why is it advantageous for tumor cells to be TAP1/TAP2 negative?
Prevents loading of peptides into MHCI, preventing lysis by CD8+ T-cells
In what way can pathogens manipulate MHC complexes to avoid immune responses?
Some pathogens downregulate MHC-complex formation or presentation on the cell surface
Which HLA-allele is most strongly associated with auto-immune disease?
HLA-DR
Variations in which part of the MHC molecule are most often associated with auto-immune disease?
Variations in the peptide-binding groove
Which T-cells are more abundant: γδ or αβ?
αβ
How are γδ T-cells activated?
MHC-independent activation by lipid antigens through CD1
How does the TCR transduce signals into the cell?
The TCR cannot transduce signals by itself and is liked to a CD3 that transduces signals
Which three signals are required for a full activation of T-cells?
- Ag-specific activation of the TCR
- Ag-independent costimulatory signal for proliferation & survival
- Cytokines that induce differentiation
What happens when a T-cell only receives a TCR signal, but not costimulatory signals and cytokines?
Induction of anergy
How can an anergic state of T-cells be overturned?
High dose of IL-2
What is the function of the Th1 subset? Against which kind of pathogens are they effective?
Cell mediated immunity, effective against viruses
Against which pathogens is the Th2 subset effective? What pathological process is it involved in?
Parasites, helminth parasites
Th2 cells are involved in allergies
Against which pathogens is the Th17 subset effective? What pathological process is it involved in?
Extracellular bacteria, fungi
Inflammation
What is the function of TFH (T-follicular helper) cells?
They are involved in the germinal centre reaction and B-cell help
What is the function of Treg cells?
Suppression and regulation of immune responses
By which cytokines is Th1 differentation induced? (i.e. what are the polarizing cytokines that will cause Th0-cells to differentiate into Th1-cells) (3)
IFN-γ
IL-12
IL-18
By which cytokine is Th2 differentation induced? (i.e. what is the polarizing cytokine that will cause Th0-cells to differentiate into Th2-cells)
IL-4
By which cytokines is Th17 differentation induced? (i.e. what are the polarizing cytokines that will cause Th0-cells to differentiate into Th7-cells) (3)
IL-6
TGF-β
IL-23
By which cytokines is TFH differentation induced? (i.e. what are the polarizing cytokines that will cause Th0-cells to differentiate into TFH-cells) (2)
IL-6
IL-21
By which cytokines is Treg differentation induced? (i.e. what are the polarizing cytokines that will cause Th0-cells to differentiate into Treg-cells) (2)
TGF-β
IL-2
What is the signature transcription factor of Th1 cells?
T-bet
What is the signature transcription factor of Th2 cells?
GATA3
What is the signature transcription factor of Th17 cells?
RORγt
What is the signature transcription factor of TFH cells?
Bcl6
What is the signature transcription factor Treg cells?
FoxP3
What are the effector cytokines of Th1 cells?
IFN-γ
IL-2
TNF-β
What are the effector cytokines of Th2 cells? (3)
IL-4
IL-5
IL-13
What are the effector cytokines of Th17 cells? (3)
IL-17A
IL-17F
IL-22
What are the effector cytokines of TFH-cells? (3)
IL-4
IL-21
IFN-γ
What are the effector cytokines of Treg cells?
TGF-β
IL-10
Different Th-cell subsets regulate each other. How do Th1 cells regulate:
Th2-cells?
Th17-cells?
Th1 cells downregulate both Th2 and Th17 cells through the release of IFN-γ
Different Th-cell subsets regulate each other. How do Th2 cells regulate:
Th1-cells?
Th17-cells?
Th2 cells downregulate both Th1 and Th17 through the release of IL-4
Different Th-cell subsets regulate each other. How do Treg cells regulate:
Th1-cells?
Th2-cells?
Treg cells downregulate both Th1 and Th2 through the release of TGF-β
What is the disadvantage of Th-subsets suppressing each other?
When the selected Th-subset is not the right one to deal with the infection, the better Th-subset is suppressed, resulting in a one-sided immune reaction
What are the two main effector mechanisms of CD8+ T-cells?
- Cytotoxicity
- Suppressing viral replication
How do CD8+ T-cells suppress viral replication?
Through the release of cytokines: IFN-γ, TNF-α, LT-α
In which two groups can CD8+ T-cell cytotoxicity be grouped?
- Calcium-dependent
- Calcium-independent
What is the calcium independent killing mechanism of CD8+ T-cells?
Release of granules containing perforin and granzyme
Perforin makes holes in the membrane, allowing granzyme to enter the cell and activate the caspase cascade, which results in apoptosis of the cell
What are the calcium-dependent killing mechanisms of CD8+ T-cells? What is the downside of this mechanism?
Ligand-receptor interactions, such as FAS-FASL
Induces caspase pathway, which results in apoptosis
Downside: only possible if the target cell has the receptor for the ligand expressed by the T-cell
What happens to effector T-cells after an infection is cleared?
Most of the T-cells undergo apoptosis, some stay behind as memory cells
In which way are memory T-cells able to mount a quicker immune response?
- Higher number of cells available in the beginning of the immune response
- Higher state of activation -> less activating signal required
How do naïve T-cells survive for a long time? What is required for this? (2)
How does this differ from memory T-cells?
Naïve T-cells survive through homeostatic proliferation
Requirements:
1. Self-antigen, presented in self-MHC
2. IL-7 + IL-15
Difference from memory T-cells: memory T-cells only require IL-7 + IL-15, but no self-peptide on self-MHC
How do memory T-cells survive for a long time? What is required for this?
How does this differ from naïve T-cells?
Memory T-cells survive through homeostatic proliferation
Requirement: IL-7 + IL-15
Difference from naïve T-cells: in addition to IL-7 and IL-15, naïve T-cells require interacting with self-peptide on self-MHC
What is the definition of a naïve T-cell? How high is their threshold of activation? Where can they be found?
T-cell that has not encountered antigen; in a resting state
Threshold of activation = high -> requires costimulatory signal
Primary location: secondary lymphoid organs
What is the definition of an effector T-cell? How high is their threshold of activation? Where can they be found?
T-cell activated by specific antigen; busy with effector functions
Threshold of activation = low -> no need for costimulation
Primary location: peripheral tissues
What is the definition of a central memory T-cell? How high is their threshold of activation? Where can they be found?
Antigen-experienced T-cell; in a resting state
Threshold of activation = low, but does require costimulation
Primary location: mostly secondary lymphoid organs, some peripheral
What is the definition of an effector memory T-cell? How high is their threshold of activation? Where can they be found?
Reactivated antigen-experienced T-cell; busy with effector functions
Threshold of activation = low -> no need for costimulation (same as regular effector T-cells)
Primary location: peripheral tissue
Which chemokine do memory T-cells home to the lymph nodes?
CCR7
How can memory T-cells be distinguished from effector memory T-cells?
Effector memory T-cells lack the CCR7-receptor that memory T-cells use to home into the lymph node
How can CCR7 be used as flow cytometry marker to analyze T-cells?
To distinguish naïve and memory T-cells (high CCR7) from effector/effector memory T-cells (low CCR7)
How can CD69 be used as flow cytometry marker to analyze T-cells?
Activation marker -> to distinguish effector T-cells (high CD69) from resting T-cells (low CD69)
How can Bcl-2 be used as flow cytometry marker to analyze T-cells?
Bcl2 is a survival signal -> to distinguish memory (very high Bcl-2)/naïve (high Bcl-2) from effector T-cells (low Bcl-2)
How can interferon-γ be used as flow cytometry marker to analyze T-cells?
IFN-γ is an effector cytokine -> to distinguish antigen-experienced T-cells (effector/memory T-cells, which have high IFN-γ) from naïve T-cells (low IFN-γ)
How can granzyme B be used as flow cytometry marker to analyze T-cells?
Granzyme B is an effector molecule in cell killing -> to distinguish effector T-cells (high granzyme B) from memory (low granzyme B)/naïve (no granzyme B) T-cells
How can CD25 be used as flow cytometry marker to analyze T-cells?
CD25 is part of the IL-2 receptor, and is an activation marker -> to distinguish resting T-cells (naïve/memory -> low CD25) from effector T-cells (high CD25)
How can CD127 be used as flow cytometry marker to analyze T-cells?
CD127 is part of the IL-7 receptor, and is a proliferation marker -> to distinguish cells that homeostatically proliferate (memory T-)cells = very high CD127 / naïve T-cells = high CD127) from effector cells (no CD127)
Why is there massive apoptosis of thymocytes in the cortex of the thymus, but rarely in the medulla?
Positive selection of T-cells takes place in the cortex; since most T-cells dont recognize peptide in the context of MHC, they will undergo apoptosis
What is specification (in the context of cellular development)?
A progenitor cell specifies to a specific lineage
Which signals are important for T-cell specification and commitment?
Notch-1: promotes T-cell development at the expense of B-cell development
Gata3/Bcl22b: commitment of T-cell fate and T-cell development
What happens when important signals for T-cell specification and commitment, such as Notch-1, Gata3 and Bcl22b are absent?
Notch-1: development of lymphoid progenitors into B-cells in the thymus
Gata3/Bcl22b: DN1/DN2-stage block
What is commitment (in the context of cellular development)?
Exclusion of fates outside the direct line of development (such as exclusion of non-T-cell fates)
What is the first process to start after commitment of a T-cell?
Rearrangement of the heavy chain (β-chain) of the TCR
What happens after rearrangement of the β/heavy chain in T-cells?
The β-chain is linked together with a PTα-chain and expressed on the surface of DN3/DN4 cells as a pre-TCR
What happens after testing of the pre-TCR on T-cells?
Cellular proliferation of cells that were activated; rearrangement of α-locus
Apoptosis of cells that failed to make a succesfull recombination
What happens to DP T-cells that are too strongly activated by peptide presented in the context of MHC?
Deletion (negative selection)
What happens to DP T-cells that are weakly activated by peptide presented in the context of MHC?
Formation of pathogen-specific CD4+/CD8+ T-cells
Which signal do DP T-cells need to be positively selected? How is a CD4+/CD8+ fate selected?
They need to recognize a peptide in the context of MHCI/MHCII. This signal will be either be enhanced by CD4 or CD8 -> determines the type of T-cell
What happens to DP T-cells that are strongly activated by peptide presented in the context of MHC (without passing the negative selection threshold)
Formation of Tregs
What happens to DP T-cells that don’t recognize peptide in the context of MHC?
Death by neglect (positive selection)
True or false: γδ-T-cells have a more diverse TCR repertoire than αβ-T-cells? Explain this.
False: γδ-T-cells have a very limited TCR repertoire because of the limited amount of V-genes available
Where do γδ-T-cells develop?
Mainly in the foetal thymus during embryogenesis
(May also form in adult thymus, but at a much lower level)
How are T-cells presented with a wide variety of auto-antigens during negative selection?
Thymic epithelial cells (mTECs) express a wide array of peripheral tissue-specific antigens (PTA’s)
There are different types of γδ-T-cells. How are they produced?
The different types of γδ-T-cells develop during different timepoints during embryogenesis, forming organ-specific subsets of γδ-T-cells
What is the positive selection of T-cells?
Positive selection takes place in the cortex, on the basis of being able to recognize peptide in the context of MHC (needed for survival)
How do γδ-T-cells recognize danger signals?
CD1 -> recognize lipids
Don’t recognize MHC and do not use CD4/CD8
What is negative selection of T-cells?
Negative selection takes place in the medulla, on the basis of autoreactivity
Which two processes are responsible for the expression of peripheral tissue antigens (PTA’s) in the thymus?
- AIRE -> transcription factor that induces expression of PTA’s in medullary TECs
- TECs mimic subsets of peripheral cells, thus expressing peripheral antigens
What is AIRE? How does it work?
AIRE = auto-immune regulator
Disrupts the 3D structure of chromatin and DNA-loops and binds to inactive promotor regions -> promotes transcriptional loops between enhancers and promotor regions -> stochastic, random expression of PTA’s by mTECs
What is meant with ‘expression of PTA’s by mTECs is mosaic’? How many mTECs express a particular PTA?
All mTECs produce a different array of antigens; all mTECs together produce all possible PTA’s
A particular PTA is only expressed by 1-3% of MTECs
Which three processes are facilitated by AIRE?
- Clonal deletion of autoreactive thymocytes -> in case of strong autoreactive signal
- Differentation of Tregs -> in case of limited autoreactive signal
- Transfer of PTA to dendritic cells, which also present PTA’s to T-cells
What does the abbreviation mTEC stand for?
Medullary thymic epithelial cell
How much of the expressed PTA in the thymus is produced through AIRE expression?
60%
Which process, other than AIRE expression, is responsible for the expression of PTA’s in the thymus? How is this achieved?
mTECs mimicing peripheral cells by expressing lineage defining transcription factors
