T cell activation and effector function Flashcards
LO
- To understand how T cells recognise antigens and how the T cell receptor is generated
- To know that T cell activation involved 3 signals: antigen, co-stimulation, and cytokines
- To know that multiple subsets of CD4 T cells exist and perform different role during immune responses
- To know the mechanism by which cytotoxic CD8 T cells kill infected cells
What are the two main types of T cells?
T helper cells= CD4
T cytotoxic cells= CD8
Tell me about the T cells and their respective MHC molecules
MHC molecules are antigen presenting cells (APC)
There are two types of MHC; MHCI and MHCII
MHCI present peptides which are recognised by CD8 cytotoxic T cells
MHCII present peptides which activate CD4 helper T cells
Whats the role of the CD4 and CD8 cells?
Soluble mediators communicate with other cells (CD4)
CD8 also involved in antigen recognition and cell-cell communication. It kills infected cells which express things they don’t usually express via peptide fragments on APC
Tell me the steps to how the CD4 T cells work
CD4
antigens recognise MHCII, antigens come from the extracellular environment, antigens get taken up by APC, macrophages, dendritic cells, and B cells are the APC. Degradation in the vesicles within cells which leads to peptide production. These peptides are loaded onto the now MHC molecules being made in the cell.
What are the three APC?
Macrophages
Dendritic cells
B cells
What are the stages to how CD8 cells work and specifically their two mechanisms
CD8
start of immune response is priming and this requires a professional APC. A dendritic cell needs to prime CD8. 2 ways.
1) pathogen can infect dendritic cell, DC starts to produce virus and viral proteins, these proteins are processed via proteolytic complex called proteasome which releases peptides which are transported to ER where they are loaded on MHCI, these MHCI are then transported to cell surface where they are recognised by CD8 T cells
2) cross presentation pathway- process takes place in professional APC, dendritic cell is the most efficient cross presentation cell because these cells can cross present antigens. Antigen present outside of DC –> taken up by DC –> degradation takes place in endosome and lysosome –> some peptides enter cytoplasm –> undergo same process as when protein is translated in cytoplasm of cells like in 1) where they go on to be loaded onto MHC molecules (involves exogenous uptake of pathogens into lysosome and endosome)
Where are the T cells developed?
Precursor cells are produced in the bone marrow, they then migrate to the thymus where they develop into T cellls
What determines whether a cell is a T cell
The expression of TCR (which recognise MHC) determines a T cell
The process in how TCR are generated are random and are not antigen determined
Tell me about the production of new T cells
Production of new T cells occurs in the foetus and the juvenile but slows down in the adult
The thymus begins to shrink after puberty
What does T cell development in the thymus involve?
Rearrangement of the T cell receptor genes to generate the diversity in the T cell repertoire (with intermediate affinity to be remaining)
Selection/ survival of T cells with a T cell receptor capable of recognised self MHC- positive selection
Removal of those T cells which express T cell receptors that bind strongly to self-antigens- negative selection
Tell me about the rearrangmenet which helps to generate the TCR genes
- TCR is comprised of alpha and beta chain
- Blue part shown in diagram is constant. These are short and don’t extend too far into cytoplasm.
- The alpha and beta heterodimer is connect via a disulphide bridge
- Variable domains recognise peptide MHC
- In alpha chain: variable domain consists of V genes (we have about 50), the J gene (we have about 70), there’s recombination signals surrounding these genes which allow enzymes to bind to recombination sequences and allow recombination of V with J, leads to juxta positioning of gene which means an V and J gene are next to one another
- In the beta chain: more diversity, there’s recombination as has V, D and J genes.
- Diversity from multi segment gene that encode antibody variable chain (recombination)
- Also get diversity from somatic hypermutation (this is not present in…)
- Can get additional AA in the junctions which are not encoded in the genome (additional diversity in this process)
- Diversity= recombination, somatic hypermutation and addition of extra AA
Explain T cell development in the thymus and how they differentiate
- precursors from bone marrow differentiate once in thymus
- DN stands for double negative as they don’t express CD4/8
- 4 stages of differentiation
- Key stages between D2 and 3 when the TCR starts to be expressed
- Pre-alpha associated with beta chain to maintain Beta chain on cell surface
- Allelic exclusion: once have one receptor on cell surface of T cell, then this cell doesn’t go through further recombination steps, ensures only one TCR per T cell
- B chain gets made first before alpha so needs to associate with pre-alpha chain
- Then starts producing the alpha chain and the two chains can come together
- Once D1–> D4 has occurred the cell then starts expressing both CD4 and CD8 cells (double positive cell, DP)
- TCR which bind MHCI down regulate CD4 to maintain CD8
- TCR which bind MHCII down regulate CD8 to maintain CD4
- This way the cells become single positive cells (CD4+ SP and CD8+ SP)
- Get positive selection at this process. If the cells are unable to recognise MHC, then they die
- In medulla (medulla epithelial cells or DC), go through negative selection process if recognise self-antigen
Tell me the first steps in TCR signalling
A single TCR/CD3 complex has 10 ITAM motifs (immune receptor tyrosine based motifs)
Phosphorylation of these ITAMs is a crucial first step in signalling
All occurs in periphery (secondary lymphoid organs)
This is performed by Src family kinases (e.g., Lck)
ITAM YXXLX7-11YXXL immunereceptor Tyrosine-based activation motif
Rough overview of TCR signalling
- Proliferation, secretion of cytokines, expression of cytotoxic molecules
- Phosphates signify the activation of T cells.
- Lck is the enzyme that is required for phosphorylation of ITAM residues
- Antigen independent way is how the CD4 and CD8 co-receptor interact with peptides
- Lck brought near TCR cytoplasmic domains
How are T cells initiated for a response?
- T cells migrate from the thymus to the blood, lymph nodes and spleen
- Antigen is most often encountered in the lymph nodes/ spleen
- Processes antigen is recognised by a specific T cell- triggers a signalling cascade
- The T cell proliferates extensively, and its progeny differentiate into effector cells (CD4 cells produce cytokine and CD8 T cells become killers)
- Effector T cells migrate to the site of infection
- A small number of cells differentiate into memory T cells
E.g., infection with EBV, about 40-50% recognise one pathogen, infection with corona, doesn’t lead to that many T cells expanding
What do dendritic cells transport?
Dendritic cells transport processed antigen for presentation to T cells
As shown in the diagram, the leaf like cells represent dying cells and some dying cells may contain virus which then is taken up by DC for APC
What are the signals that are involved in T cell activation?
Signal 1: peptide/ MHC: TCR
Signal 2: co-stimulation (induced by innate immunity)- antigen independent, signal 1 + signal 2 leads to expansion of T cells
Signal 3: cytokines (induced by innate immunity)- help to differentiate the naïve T cells
What is required for a successful T cell response?
Dendritic cell activation
Tell me about dendritic cell maturation
DC present antigens to T cells
In an immature state, then become mature by responding to innate signals
Immatures are good are taking up pathogens for phagocytosis
Once mature, they can’t take up more antigens for, instead they become better at stimulating T cells and become more immunogenic
Explain how the activating signals of the T cells work together
1 cannot proliferate very well only once 1 and 2
2 is delivered by signal CD28 which binds to B7 which is expressed on APC
B7 is upregulated because of the innate immune system
IL-2 and upregulation of IL-2R are due to signal 1 and 2
Differentiation cytokines (3), these controls T cell function. List of some of the cytokines produced are above.
Differentiate into effector cell which can mediate the anti-viral immune response
Tell me about co-stimulation and the families which are involved in this
Also known as signal 2
Synergise with TCR signal 1 signalling
Required for most T cell responses in vivo
Two major families:
- Ig superfamily: CD28 (first co-stimulatory molecule discovered) and ICOS
- TNFR superfamily: CD27, OX40, 4-1 BB
Tell me about CD28 and its main roles, its ligands and explain what could happen if one is deficient in CD28
Main role: proliferation, survival, and IL-2 production
Ligands: CD80 and CD86 expressed constitutively at low levels but rapidly induced on APCs
CD28 deficient mice: defects in CD4 and CD8 responses to microbes (CD4>CD8). Defect in CD4 T cell help for B cells (germinal centre, Ab class switching)
Tell me about ICOS and ICOSL
ICOS
Main role: generation of Tfh cells that regulate germinal centre reactions and humoral immunity
ICOS deficient humans and mice: few Tfh and defect in germinal centre formation, Ab class switching and memory B cells
CD28 vs ICOS
Tell me about the negative regulation of APC with CTLA-4
CTLA-4 is similar to CD28 but doesn’t signal stimulation. It is an inhibitory molecule (helps with negative feedback)
B71 and B72 are both co-stimulatory molecules?
Auto-immune disease and inflammation found in rats with excessive proliferation due to absence of CTLA-4
CD28 and CTLA-4
What does CTLA-4 blockade promote?
anti-tumour T cell immunity
What do naïve CD4+ T helper cells differentiate into?
effector cells with different functions
What are the different effector cells that naïve CD4 cells can differentiate into?
Th1
Th2
Th17
Tfh
With the effector cells which naïve CD4+ T helper cells differentiate into, explain their roles and the types of cytokines they secrete
- T cell subsets can deal with certain types of pathogens
- Naïve CD4 T cells can go towards any subset
- Signal 3 cytokines are produced due to infection and can help drive the differentiation of CD4 naïve cell to any subset
- All subsets are types of CD4 cells
How do T follicular helper T cells (Tfh) promote the humoural (antibody) response?
- B cell proliferation/ survival
- B cell differentiation into plasma cells (antibody secreting cells)
- Antibody class switching IgM –> IgG, IgA or IgE
- Affinity maturation to generate high affinity antibodies
- Memory B cell generation
Whats the Tfh role in the lymph node?
In lymph node the B and T cell occupy different zones
CD4 need to switch of CCR7 and switch on CXCR5 which is the receptor for CXCL13 (produced in follicle), this allows Tfh to move into B cell zone and helps B cells undergo germinal centre reaction (somatic hypermutation to produce antibodies with high affinity for pathogen), also helps prolonged life of antibodies
CD4+ T cell help for B cells
Explain Th1 differentiation
- Antigen and IFN-gamma from innate cells upregulate T-bet expression
- T-bet increases the expression of the IL-12 receptor beta2
- IL-12 from the dendritic cells drive differentiation into Th1
- Differentiated Th1 cells can be induced to produce IFN-gamma by either TCR
- IFNgamma is used for activating interfering macrophages
- NK cells produces IFNgamma
- Early IFNgamma and IL-12 is needed for differentiation to Th1 cells
- Th1 main function is to produce IFNgamma for activation of macrophages
- stimulation or exposure to the cytokines IL-12 and IL-18
Whats the link between IFNgamma with B cells and macrophages
Th1 cells summary
mediators of antimicrobial and cytotoxic activity of macrophages and neutrophils
What are the ways in which Th2 cells differentiate?
- Deal with extracellular pathogens
- Cytokines induced by parasitic infections e.g., helminths
- Key differentiation cytokines
IL-4
Thymic stomal lymphopoietin (TSLP)
IL-25
IL-33
- Key cytokine produced by TH2 cells
IL-4
IL-5
IL-13
What is the principal switch factor for production of IgE by B cells?
IL-4
What does IgE bind to?
What does their binding cause?
IgE binds to specific receptors on mast cells, basophils, and eosiniphils. T
he binding of pathogens to IgE causes the release of inflammatory mediators, reactive oxygen intermediates and anti-microbial peptides
What are IL-5 activated eosiniphils effective in?
Immune responses against extracellular parasites e.g., nematode worms
What do eosinophils secrete and what are these involved in?
Eosinophils secrete and array of cytotoxic granule cationic proteins (e.g., major basic protein, eosinophil cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin) that are capable of inducing apoptosis and necrosis in target cells
What does the cytokine IL-13 induce?
IL-13 induce an increase in goblet cell number and mucus production which help repel intestinal worms. It also increases contractility of intestinal longitudinal smooth muscle
What is the role of IL17 and IL-7F?
IL17 and IL-17F
Increase pro-inflammatory cytokines (IL-6, TFNalpha and IL-1beta) and chemokines
Increase neutrophil recruitment
Tell me about IL-22 i.e., targets, what it maintains and induces
IL-22
Target cells of epithelial origin
Maintains integrity and barrier function in the small intestine and skin
Induces expressed of beta-defensins (antimicrobial peptides) in epithelial cells
Tell me the following about CD8+ cytotoxic T cells
What they recognise
Their main function
Their differentiation
Recognise MHCI presented by all nucleated cells in the body
The main function of cytotoxic T cells is to kill cells that are infected with intracellular pathogens e.g., virus infected cells
Cytotoxic T cells are also capable of killing cancer cells which express mutated proteins
Naïve CD8 T cells differentiate into cytotoxic cells after activation by antigen, co-stimulation, and cytokines (IL-2 and IL-12)
Cytotoxic T cells are serial killers
What is the mechanism of cell killing by cytotoxic T cells
express molecules (which are help in secretory vesicles as are so toxic e.g., secretory lysosome)
Granzymes and perforins are molecules help with killing and are what is found in secretory lysosomes
Reorientation of microtubule organising centre (centrosome which is used by cells in cell division to form polar end of spindle), these centrosomes (made of two centrioles) are initiated. These move into contact zone (immunological synapse) between T cell and MHC. This zone is also where growth of microtubule occurs
Secretory lysosome is reeled onto contact zone, the killing site
Prevent killing of the bystanding cells within the area of the infected cell
How do cytotoxic T cells deliver their cytotoxic proteins?
In a highly polarised manner
Focused attack- re-orientation of the microtubule organising centre (MTOC) from which microtubules grow
Lytic granules move along the microtubules towards the MTOC
Prevent killing of ‘innocent bystander’ cells that are not infected
Tell me about perforin
Pore forming protein via polymerisation
Perforin gene KO mice are profoundly immunodeficient- susceptible to viral infection and cancer
Delivers granzymes to the target cell which leads to cell death
During biogenesis and storage, how does the cell protect it self from perforin?
Binds to inhibitors, calreticulin and serglycin and is unable to oligomerise
Inactive in the acidic environment of the granule’s pH 5.1-5.4
Requires proteolytic cleavage by a cysteine protease to be active (removes carboxy-terminal peptide)
How are granzymes delivered into cells?
via pores which the perforin has made in target cells
What are the most abundant granzymes?
Granzyme A and B are the most abundant granzymes
What type of molecule are granzymes?
Granzymes are a type of serine protease
How are granzymes produced?
As inactive pro-enzymes and converted into active enzymes by Cathepsin C
When are granzymes inactive?
At the acidic pH of the granules
What type of complex do granzymes form?
Form large (200nm) complex with serglycine (proteoglycan)
Tell me the steps to granzyme B’s function
Activate programme cell death pathway in target cell
Caspase 3 is an executioner caspase
All caspases are produced in an inactive form and granzyme B cleaves them into their active form which then leads to cell death
CAD associates with inhibitor, CAD
Cytochrome C is a signal which activates cell death
Activation of caspase 9 –> activation caspase-3 which is the end product caspase
Tell me what granzyme A’s function is
Induces cell death synergistically with granzyme B
Triggers death by a different mechanism to granzyme B
Granzyme A activates the DNase NM23-H1, an enzyme that causes DNA nicks, by destroying its inhibitor, the SET complex (physiological function: transcription/ transcription-related DNA repair)
Granzyme A also targets the linker histone H1 which leads to chromatin opening thus allowing access to the DNA by other nucleases
It also cuts lamins, the main structural components of the nuclear envelope
What are the mechanisms of killing cytotoxic T cells?
Provide self-protection- active cathepsin B is present on the surface of cytotoxic T cells following granule release. Cathepsin B degrades membrane inserted perforin in the T cells
Prevent the killing of DCs- expression of serine protease inhibitors (serpins) that prevent the function of granzyme B
What type of T cell are regulatory T cells (T Regs)?
They are a type of CD4 cell
What are the two types of regulatory T cells?
Thymic and peripheral
What is the role of the regulatory T cells?
Inhibit T cell responses against self- dietary and commensal antigens
Protect against autoimmunity
Protect host by inhibiting other T cells
Why aren’t T reg cells removed from the body?
T cell reg have a higher affinity for peptide MHC than normal CD4 T cells. But not enough to be deleted in the thymus
What type of cells are regulatory T cells?
suppressive cells
What do T reg cells compete for?
Compete for self-reactive/ autoreactive T cells, produce mediators that suppress other cells and repress autoimmune diseases
T reg cells
