1
Q
What is flow cytometry used for?
A
To count and analyse the size, shape and properties of individual cells within a heterogeneous population of cells
2
Q
What type of data is in flow cytometry?
A
Quantatative
3
Q
What is analysed in a flow cytometer?
A
- Cells
- Tissues
- Organisms
4
Q
What is the flow order of a flow cytometer?
A
- Sample
- Fluidics that move the sample into the flow cytometer
- Lasers
- Optics which gather the light
- Detectors to sense the light
- Computer system to output the data into a form that can be analysed
5
Q
What is the forward scatter proportional to in a flow cytometer?
A
The size of the cell
6
Q
What occurs to the forward (side) scattered light in a flow cytometer?
A
- Detector converts the scattered light into a voltage which is directly proportional to the amount of forward scattered light
- The computer converts this data into a histogram plot with amount of forward scattered light on the x-axis and the number of cells on the y-axis
7
Q
What is the side scattered light proportional to in a flow cytometer?
A
Shape and internal complexity of a cell
8
Q
What form does the data of fluorescence take?
A
- Histogram plot
* Dot plot
9
Q
What are the uses of antibodies?
A
• Blocking/neutralising o Receptors o Cytokines o Pathogens o Toxins • Capturing o Precipitation o ELISA o Purification of cells • Immunotherapy • Scientific and clinical study
10
Q
What is immunodiagnostics?
A
Diagnostic methodology that uses antigen-antibody reaction as their primary means of detection
11
Q
What can be used to link as a label with an antibody?
A
- Radiation
- Enzyme
- Fluorescent tag
12
Q
What diagnostic methods can use labelling?
A
- Western blotting (antigen-enzyme)
- Immunohistochemistry
- Flow cytometry (antigen-fluoresce)
13
Q
What does CD number stand for?
A
Cluster of differentiation number
14
Q
What are CD numbers used for?
A
Used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells
15
Q
What does CD4- mean?
A
The patient does not have CD4 cells
16
Q
What does CD4+ mean?
A
The patient has CD4 cells
17
Q
What does CD16low mean?
A
The patient has a low concentration of CD16
18
Q
What does CD16high mean?
A
The patient has a high concentration of CD16
19
Q
On a histogram where is the positive and negative CD numbers shown?
A
- Negative to the left
* Positive to the right
20
Q
What diseases are due to too much immune reactivity?
A
• Autoimmune diseases o Rheumatoid arthritis o Multiple sclerosis o Lupus • Atopic diseases o Asthma o Siriasis o Anaphylaxis
21
Q
What diseases are due to too little immune reactivity?
A
- Infection
* Cancer
22
Q
What is the definition of tolerance?
A
- Teaches what is “self” allowing the adaptive immune system to recognise “non-self”
- Essential to prevent autoimmune disease
- Immunocompetent host fails to respond to an immunogenic challenge with a specific antigen
23
Q
Who discovered that tolerance is acquired and specific?
A
Peter Medewar by doing skin grafts on mice
24
Q
What is the danger hypothesis?
A
If encounter an antigen in a safe environment, the body is tolerant
• If encounter an antigen in a dangerous environment, the body is not tolerant
25
What is central tolerance?
* Immature self-reactive lymphocytes that recognise self-antigens in generative (“central”) lymphoid organs die by apoptosis; other fates
* This occurs during lymphocyte development
26
What is peripheral tolerance?
* Mature self-reactive lymphocytes that recognise self-antigens in peripheral tissues are inactivated (anergy), killed (deletion) or supressed
* Occurs after lymphocytes leave the primary lymphoid organs
27
What features does tolerance have the same as an immune response?
* Antigen specific
* Can exist in T cells, B cells or both
* Has immunological memory (T cell tolerance is more profound and lasts longer then tolerance at the B cell level)
28
Where do B cells originate from?
Bone marrow
29
Where do T cells originate from?
Thymus
30
What does kinetic proofreading do?
* Measure of affinity
| * Assesses how well the T cell receptor and its target antigen is bound
31
What does a high affinity in kinetic proofreading mean?
Strong binding which lasts between T cell receptor and its target antigen
32
What does a low affinity in kinetic proofreading mean?
Weak binding which falls apart between T cell receptor and its target antigen
33
What does a resting T cell receptor in a cell membrane that is unphosphorylated mean?
That the T cell receptor is turned off
34
What does Lck want to do to a T cell receptor?
Phosphorylate TCR
35
What does CD45 want to do to a T cell receptor?
Dephosphorylate TCR
36
What does ZAP70 do to a T cell receptor?
Binds to phosphorylated TCRs, signals TCR activation to the rest of the cell
37
What occurs on the cell membrane of a T cell with regards to a resting TCR?
Lck and CD45 compete = no overall receptor phosphorylation and no ZAP70 binding
38
What occurs on the cell membrane of a T cell with regards to the TCR and an irrelevant target antigen?
* No strong interaction = no overall activation
| * No ZAP70 binding
39
What occurs on the cell membrane of a T cell with regards to the TCR and a specific target antigen?
* Strong/close interaction = overall activation
* CD45 is too big and is excluded
* Lck is small and can still get to phosphorylate TCR
* ZAP70 binding = T cell becomes activated
40
What is an immature T cell called?
Thymocyte
41
What question must be asked before a thymocyte can be released?
* Does it see self MHC at all?
* Does it see self MHC too much?
* Does it react with self-peptides?
* Will it fight intracellular or extracellular infections?
42
Why is a mechanism for repertoire selection and self-tolerance needed?
* Generation of the TCR repertoire involves many random mechanisms
* The specificity of TCR in the immature repertoire is also random and will include cells with receptors that are harmful (self-antigen recognition), useless and useful (foreign antigen recognition)
43
Do processing pathways distinguish between self and non-self?
No
44
What type of antigen is most presented by an antigen presenting cell?
Self-antigens
45
What is the thymus?
* Lobulated structure with a stroma of epithelial cells and connective tissue
* Stroma provides microenvironment for T cell development and selection
* Lobules differentiate into an outer cortex and inner medulla, both filled with bone-marrow-derived thymocytes
46
What cells are in the cortex of the thymus?
* Thymocyte
* Cortical epithelial cell
* Immature double positive or double negative thymocytes
47
What cells are in the medulla of the thymus?
* Thymocyte
* Dendritic cell
* Macrophages
* Medullary epithelial cells
* Mature single positive or single negative thymocytes
48
How are harmful T cells removed?
Negative selection
49
How are useless T cells removed?
Neglect - no signal sent to keep it alive
50
What selection occurs to useful T cells?
Positive selection
51
What is required for T cell maturation?
Thymus
52
What happens to T cells if the thymus is removed from an adult and a neonate?
* Adult will have mature T cells within its body - we need the thymus to develop/mature T cells not to maintain them – bone marrow supplies T cells and they mature in the thymus
* Neonates will have no T cells as the T cells have not yet left the thymus in neonates
53
What percentage of T cells will die in the thymus as a result of stringent processing to find the "useful" T cells?
Approximately 98%
54
What gets rid of the apoptotic thymocytes?
Thymic macrophages
55
What is the flow order of T cell development?
* Double negative
* Large double positive
* Small double positive
* Either single positive (with CD4) or single positive (with CD8)
56
What are the TCR associated molecules and coreceptors of double negative T cells?
* CD3/TCR-
| * CD4-/CD8-
57
What are the TCR associated molecules and coreceptors of large double positive T cells?
* CD3+
* TCR+
* CD4+/CD8+
58
What are the TCR associated molecules and coreceptors of small double positive T cells?
* CD3+
* TCR+
* CD4+
* CD8+
59
What are the TCR associated molecules and coreceptors of single positive T cells (with CD4+)?
* CD3+
* TCR+
* CD4+
60
What are the TCR assocaited molecules and coreceptors of single positive T cells (with CD8+)?
* CD3+
* TCR+
* CD8+
61
How does the thymus choose which of the cells entering the thymus are useful, harmful or useless?
* Goes through both positive and negative selection
| * If lives through both it is useful
62
What is positive selection?
Retention of thymocytes expressing TCR that are restricted in their recognition of antigen by self MHC i.e. selection of the useful
63
What is negative selection?
Removal of thymocytes expressing TCR that either recognise self-antigens presented by self MHC or that have no affinity for self MHC i.e. selection of the harmful and the useless
64
What is MHC restriction?
* Antigen can be seen by the TCR only in the context of an MHC molecule
* TCR will not bind to an MHC molecule unless there is an antigen in the groove
* In the presence of antigen, the TCR must have some affinity for the MHC molecule
65
What do studies in bone marrow chimeras show in the thymus?
MHC restriction is learnt in the thymus
66
What MHC haplotype will T cells recognise in bone marrow chimera experiment?
* Mice of a particular MHC haplotype only make T cells restricted by that haplotype
* Bone marrow must contain potential to make T cells restricted by A and B MHC molecules (cross)
67
What does irradiation prevent in bone marrow chimera?
* Prevents the bone marrow from generating lymphocytes
| * Irradiation destroys the immune system but has no effect on the epithelial or dendritic cells of the thymus
68
How is MHC restriction in the thymus learned?
By positive selection
69
What cells are restricted by MHC type of the thymus that they mature in, in bone marrow chimera?
Peripheral T cells
70
How is neglect achieved in useless thymocyte cells?
* Peptide is not recognised or irrelevant
| * Thymocyte receives no signal, fails to be positively selected and dies by apoptosis
71
How is positive selection achieved in useful thymocyte cells?
* Peptide is a partial agonist
* Thymocyte receives a partial signal and is rescued from apoptosis i.e. the cell is positively selected to survive and mature
72
How is negative selection achieved in harmful thymocyte cells?
* Peptide is an agonist
| * Thymocyte receives a powerful (full) signal and undergoes apoptosis i.e. the cell is negatively selected and dies
73
How accurate is the positive selection model?
* Relied on very complex chimera experiments
* Relied on proof of MHC restriction as an outcome which is tested in an ‘unnatural’ response using MHC mismatched presenting cells
74
How accurate is the negative selection model?
Relied on exceptionally powerful superantigens operating outside the normal mechanisms of antigen recognition
75
In a TCR from MHC class I restricted T cell, which type of cells mature?
CD8
76
In a TCR from MHC class II restricted T cell, which type of cells mature?
CD4
77
What are the models for double positive to single positive transition?
* Instructive model
| * Stochastic/selection model
78
What is the instructive model?
Signal from CD4 silences the CD8 expression and vice versa
79
What is the stochastic/selection model?
Cells randomly inactivate CD4 or CD8 gene, whilst testing a match of TcR restriction
80
What is the differential effect on the mature and immature repertoire in deletion of cells in the thymus?
* TcR from T cell specific for hen egg lysosome (HEL)
* Approximately 100% of T cells express anti-HEL TcR
* Thymocytes activated by antigen in the thymic environment die
* T cells activated by antigen in the periphery proliferate = evidence for negative selection
81
What occurs to thymocytes in negative selection when in the presence of ecotopic expression of host peptides?
Development of regulatory T cells
82
What are the consequences of AIRE mutation?
* Human disease
| * Mouse knockout
83
What human disease is a result of AIRE mutation?
* Autoimmune polyendocrinopathy with candidiasis and ectodermal dysplasia (APECED, also called autoimmune polyendocrine syndrome (APSS-1)
* Associated gene identified by positional cloning, named AIRE (“autoimmune regulator”)
84
What is mouse knockout?
* Autoantibodies against multiple endocrine organs, retina
| * Failure to express many self-antigens in the thymus -> failure of negative selection
85
How are self-peptides presented in the thymus?
* AIRE (autoimmune regulator) is a regulator of gene transcription that stimulates thymic expression of many self-antigens which are largely restricted to peripheral tissues
* Discovered as the genetic cause of a human autoimmune disease APS-1
86
Summarise positive selection
* Bone marrow chimeras show that MHC restriction is learnt in the thymus
* T cells are ‘educated’ in the thymus to recognise antigens only in the context of self MHC
* MHC restriction is learnt in the thymus by positive selection
* The MHC haplotype of the environment in which T cells mature determines their MHC restriction element
87
Summarise negative selection
• Two different models can explain the choice whether to become CD4 or CD8 single positive T cells
• AIRE causes thymic epithelial cells to produce many self-proteins
o But not proteins that are in immune privileged sites
o These are captured and presented by professional APCs in the thymic medulla
• Mutations in AIRE lead to failure in negative selection and profound autoimmunity
88
What is the significance of tolerance?
* All individuals are tolerant of their own antigens (self-tolerance); breakdown of self-tolerance results in autoimmunity
* Therapeutic potential
89
What is the therapeutic potential of tolerance?
Inducing tolerance may be exploited to prevent graft rejection, treat autoimmunity and allergic diseases, and prevent immune responses in gene therapy, perhaps stem cell transplantation
90
What is "clonal ignorance"?
Mature self-reactive lymphocyte clones do not encounter or respond to self-antigens
91
What does anergy in peripheral tolerance result in?
Functional unresponsiveness as anergy inactivates T cells by turning off signals with APCs
92
What does deletion in peripheral tolerance result in?
Apoptosis after activating cell with APCs
93
What does suppression in peripheral tolerance result in?
Block in activation by regulatory T cells between APCs and T cells
94
What does T cell anergy in peripheral tolerance do?
Either blocks signal or engages inhibitory receptors (e.g. CTLA-4)
95
What are the features of anergy in peripheral tolerance?
* Induced by self-antigens that are displayed to the immune system without inflammation or innate immune response (prolonged signal 1, i.e. antigen, inadequate signal 2)
* Recognition of such antigens may lead to signalling block and/or engagement of inhibitory receptors
* Role of anergy in self-tolerance in humans is unclear (therapeutic potential?)
96
What does deletion in peripheral tolerance do?
Either activation induced cell death (engagement of death receptors) or activation induced cell death (expression of pro-apoptotic proteins) both ending in apoptosis
97
What are the features of deletion in peripheral tolerance?
Stimulation of T cells by self-antigen triggers apoptosis by engagement of death receptors (death receptor pathway) or imbalanced expression of pro-apoptotic proteins (mitochondrial pathway)
98
What evidence is there to support the importance of ACID in maintenance of self-tolerance?
* Mice with mutations in Fas or Fas ligand develop a lupus-like autoimmune disease
* Humans with mutations in Fas or enzyme involved death receptor-induced apoptosis (caspases): the autoimmune lymphoproliferative syndrome (ALPS)
* Eliminating both death pathways in mice -> “spontaneous” systemic autoimmune disease
99
What does suppression in peripheral tolerance do?
* Recognition of self-antigen in either thymus or peripheral tissues by regulatory T cells
* Inhibit T cell activation (dendritic cell with naïve T cell) or inhibit T cell effector functions
100
What are the features of suppression in peripheral tolerance?
* Regulatory T cells are CD4+ cells that express high levels of CD25 (IL-2 receptor alpha chain)
* Generated by self-antigen recognition in the thymus or peripheral tissues
* Generation requires a transcription factor called Foxp3 (mutations in Foxp3 are the cause of a severe autoimmune disease in humans and mice
101
What are the mechanisms of action in suppression in peripheral tolerance?
* May be multiple
* Secretion of immune-suppressive cytokines (IL-10, TGFβ) – inhibits macrophages, dendritic cells and interferon gamma
* CTLA-4 on Tregs blocks B7 on APCs
102
What is the significance of suppression in peripheral tolerance with regards to self-tolerance?
* Some autoimmune diseases may be associated with defects in regulatory T cells or resistance of responding cells to suppression
* Therapeutic potential of cellular therapy (autoimmune diseases, graft rejection etc)
103
What is the largest immunologic organ in the body?
The gastrointestinal tract
104
In the GIT what might take up antigens in oral tolerance?
* Peyer’s patches
* Dendritic cells
* Epithelial cells
105
What is the most important immune cell in oral tolerance induction?
Regulatory T cell
106
What factors can influence oral tolerance induction?
* Antigen related
* Inherent to the host
* Disturbances at different steps can be described in food hypersensitivity
107
The introduction of antigens to the body has different routes with distinct outcomes. What routes lead to immunity?
* Subcutaneous, intramuscular and intradermal
| * Infection/injury
108
The introduction of antigens to the body has different routes with distinct outcomes. What routes lead to tolerance?
* Intravenous
* Portal vein
* Anterior chamber of the eye
* Mucosal
109
What can oral feeding of antigen induce?
An active (but inhibitory) immune response that is mediated by T cells
110
What factors allow oral tolerance to be multifactorial?
* Induction of clonal anergy
* Induction of clonal deletion
* Induction of T regulatory cells which produce suppressor cytokines
111
What does a low dose oral tolerance mean?
Immunoregulation – some immune cells prevent others from responding to antigen
112
What does a high dose oral tolerance mean?
Anergy/deletion – the cells themselves lose the ability to respond to the antigen used to induce oral tolerance
113
Compare use of antigen between oral tolerance and vaccines?
* Tolerance has a soluble antigen
| * Vaccines have an antigen plus adjuvant, particulate antigens
114
Compare number of dosages between oral tolerance and vaccines?
* Tolerance has repeated doses (prolonged and sustained)
| * Vaccines have a limited number of vaccine doses
115
Compare amount of dosage between oral tolerance and vaccines?
* Bolus dose of 20-500mg or multiple injections of 1mg
| * Vaccines have a low antigen dose, typically in ug concentrations
116
What are some types of viral infections?
* Encephalitis/meningitis
* Eye infection
* Common cold
* Parotitis
* Pharyngitis
* Gingivostomatitis
* Cardiovascular
* Pneumonia
* Myelitis
* Hepatitis
* Pancreatitis
* Gastroenteritis
* Skin infections
* Sexually transmitted diseases
117
What diseases are linked (not all fully confirmed) to infection?
* Alzheimer’s disease
* Asthma
* Atherosclerosis
* Autoimmune diseases
* Cancer coronary heart disease dementia
* Stroke etc
118
What autoimmune diseases are linked (not all fully confirmed) to infections?
* Crohn’s
* Lupus
* Multiple sclerosis
* Psoriasis etc
119
What does Epstein Barr Virus cause?
* Autoimmune diseases
* Breast cancer
* Oesophageal cancer
* Hodgkin’s lymphoma
* Chronic obstructive pulmonary disease
* Seasonal affective disorder
* Lupus multiple sclerosis
120
How many amino acids is the class I epitopes of antigens?
8-10
121
How many amino acids is the class II epitopes of antigens?
18-20
122
What are some features of viruses?
* Simple structure (subcellular level)
* Obligate intracellular agents – unable to replicate outside cells, enter cells via receptors
* Induce diseases through damage of cell in which they replicate or induction of the immune response
* Infection can be either acute or chronic (active and latent)
123
What are the mechanisms of the innate immunity as a defence against viruses?
* Inhibition of infection and induction of antiviral state type I interferons (IFN-α and β)
* Killing of infected cells (NK cells)
124
How is antiviral action of type I interferons done?
Once a cell is infected with a virus it will secrete type I interferons which leads to the upregulation of enzymes that inhibit replication (non-specific) of uninfected cells with IFN receptors protecting it from infection. The type I interferons also increase expression of class I MHC molecules which promotes cytotoxic T cells to kill the infected cell
125
What happens in the destruction of infected cells by NK cells when the inhibitory receptor is engaged?
NK cell binds to a normal autologous cell by the activated receptor to the ligand for NK cell and the inhibitory receptor that binds to the self-class I MHC – self peptide complex. This leads to the NK cell not activating so the normal autologous cell is not killed
126
What happens in the destruction of infected cells by NK cells when the inhibitory receptor is not engaged?
NK cell only binds to the ligand for NK cell of the virus infected cell (class I MHC negative – virus inhibits class I MHC expression). the NK cells are activated and kills the infected cell
127
What are the mechanisms of the adaptive immunity as a defence against viruses?
• Humoral immunity – B cells and antibody lead
o Neutralization (IgG and IgA)
o Antibody-dependent cell mediated cytotoxicity (IgG)
o Opsonization (IgG)
• Cell mediated immunity – CD8+ and CD4+ T cells lead
o Killing of infected cells (CD8+ T cells)
o Activation of CD8+ T cells and B cells (CD4+ helper T cells)
128
How does neutralization of viruses occur without antibodies present?
* No neutralization
* Microbe can penetrate the cells to the epithelial cells where it can bind to tissue cell receptor and infect the tissue cell
129
How does neutralization of viruses occur with antibodies present?
* Neutralization
| * Antibody blocks binding of microbe to the cells so they cannot bind to the cell and infect them
130
What are the protective mechanisms of antibodies?
* An antibody can bind to the virus and C1q can bin to the antibody. This activates complement with complement components binding to complement receptors on the macrophage allowing phagocytosis
* An antibody can bind to the Fc receptor on a macrophage blocking the binding of the virus to that Fc receptor allowing phagocytosis
* Antibodies can bind to the virus blocking the uptake through cellular receptors on target cells
* Antibodies can signal to NK cells that they have bound to a virus on a (infected) target cell. The NK cell then kills the target cell
131
What is the mechanism of killing by CTLs (cytotoxic leukocytes)
* Antigen recognition and conjugate formation
* CTL activation
* CTL granule exocytosis
* Apoptosis of target cell
* Granzymes enter cell via receptor-mediated endocytosis and enter cytoplasm via perforin-dependent mechanisms -> activation of apoptotic pathways
132
How does an NK cell kill?
* Either by perforin and granzymes resulting in apoptosis
| * Or FasL and Fas receptor binding resulting in apoptosis
133
What are some mechanisms of immune evasion of viruses?
* Antigenic variation (influenza, HIV …)
* Inhibition of antigen processing and presentation (many viruses)
* Inhibition of immune response (many viruses)
* Infection of immune cells (HIV…)
* Establishment of latency (HSV, HIV…)
* Inhibition of apoptosis (Herpes and Pox viruses …)
134
How has the Influenza virus gone through antigenic variation?
* Influenza infected humans and birds
| * An epidemic strain that affects both birds and humans then infect a pig with a pandemic strain
135
How does a virus inhibit antigen processing and presentation?
• They can inhibit the class I MHC pathway of antigen processing
o They inhibit the proteasomal activity (done by EBV and human CMV)
o Block the TAP transport (HSV)
o Block MHC synthesis and/or ER retention (adenovirus or human CMV)
o Removal of class I from ER (CMV)
o Interference with CTL recognition by “decoy” vital class I-like molecules (murine CMV)
136
How can viruses inhibit immune responses?
Through production of virokines and viroreceptors
137
How does Epstein Barr Virus inhibit the immune response?
EBV infected B lymphocyte inhibits macrophage activation by secreting IL-10 and binding to the receptor on the macrophage inhibiting it
138
How does Poxx Virus inhibit the immune response of the body?
• Pox virus infected tissue produces IL-1 and IFN-γ receptors (viroreceptors) to block IL-1 and IFN- γ activation on the macrophage
139
What is a potential disadvantage of antigenic variation?
Requires mutation
140
What are the benefits of antigenic variation?
* Extend the length of infection
| * Infect hosts with prior exposure
141
Why is an extended length of infection a benefit of antigenic variation?
* The new variants allow the virus to stay one step ahead of the immune response as with each new mutant strain the host must generate a new response - Use high mutation rates to stay ahead of the adaptive immune response
* Some viruses, like hepatitis C virus, escape by evading antibody response or a persistent infection with a high variability in the envelope protein indicating positive selection (that the infected cell in useful)
* Use antigenic variation to escape surveillance (Trypanosoma brucei achieves this not by mutation but by differential expression)
* Some viruses persist in vivo by ceasing to replicate until immunity wanes (latency – not transcriptionally active, and causes no disease)
142
How is antigenic variation achieved?
* Mutation
* Recombination
* Differential expression of archived variants
* HIV evades CTL responses by changing their dominant epitopes
* Epitopic space allows for a higher capacity for infection
143
How does CTLs kill in a normal immune response?
* MHC molecules present antigen from cytosol
| * Cytotoxic T cell recognises complex of viral peptide with MHC class I and kills infected cell
144
What peptides does class I present?
Cell made intracellular peptides
145
What peptides does class II present?
Found exogenous peptides from the environement
146
How do viruses escape CTLs?
Amino acid substitutions of the epitopes
147
How can amino acid substitutions of the epitopes allow them to escape recognition?
* Can interfere with processing and transport of peptides
* Can reduce binding to MHC molecules
* Can reduce the affinity of TCR receptor binding
148
How can CTL escape be done by interfering with processing/transport?
A study of murine leukaemia virus (MuLV – oncogenic retrovirus) showed that a single amino acid substitution in a viral peptide can alter cleavage pattern and hence epitope presentation and hence CTL response
149
What type of mutation is the single amino acid substitution responsible for CTL escape?
Single point mutations
150
What is the end result of interfering with processing/transport in CTL escape?
The epitope is unavailable to MHC and CTL responses to FMR type is weak
151
How can CTL escape be done by reducing MHC binding?
* Mutations that reduce peptide-MHC binding
* This can either prevent MHC from dragging the peptide successfully to the cell surface, or from holding on to it once there
152
What is an example of reduced MHC binding for CTL escape?
• SIV/macaques used as a model for HIV
o An epitope Tat was shown to have mutation in the Tat viral epitope that destroyed binding to the MHC
o Little variation outside of the epitope
o End result - Positive selection to block MHC binding
153
What is an example of reduced TCR binding to escape CTLs?
* LCMV system shows a single amino acid change that can lead to a decline in affinity for the TCR
* Tissot et al (2000) showed a Y to F substitution in one immunodominant epitope caused a 100-fold reduction in affinity for the TCR
* End result – escape mutation that destroy the immune system’s ability to see that viral epitope
154
What are some examples of pathogens that sample from a pool of archival genomic variation?
* Trypanosoma brucei
* Borrelia hermsii
* Plasmodium falciparum
155
What does Trypanosoma brucei cause?
Sleeping sickness
156
What does Borrelia hermsii cause?
Relapsing fever
157
What does Plasmodium falciparum cause?
Malaria
158
Why do sensory neurons remain infected?
* The virus remains quiescent, few viral proteins are produced and hence there are few virus-derived proteins to present on MHC class I
* Neurons carry low levels of MHC class I molecules making it harder for CTLs to recognise and kill them
159
Why would neurons have low MHC I expression?
Low levels of MHC I expression may be beneficial to the host since it reduces the risk that neurons, which cannot regenerate, will be attacked inappropriately by CTLs
160
How does Mycobacterium tuberculosis use antigenic variation to extend the length of infection?
* Resists destruction b host defence mechanisms/exploits them
* It is taken up by macrophages but prevents the fusion of the phagosome with the lysosome, effectively hiding from antibody-mediated immunity
161
What vaccine is a good vaccine in that is has excellent cross-reactivity?
Measles virus vaccine
162
When does cross-reaction occur?
Occurs when the host can use its specific recognition from prior exposure to fight against a later, slightly different antigenic variant
163
What causes antigenic drift?
Point mutations in the genes encoding surface proteins
164
What causes antigenic shift?
Reassortments leading to novel surface proteins
165
What are features of antigenic drift?
* Seasonal (Influenza)
* Predictable annual occurrence
* Residual immunity in population
* Neutralizing antibodies against hemagglutinin/neuraminidase block binding to cells
* Mutations alter hemagglutinin/neuraminidase epitopes so that neutralizing antibody no longer binds
166
What are features of antigenic shift?
* Pandemic (Influenza)
* Unpredictable rare occurrence
* “Naïve” population
* Antigenic shift occurs when RNA segments are exchanged between viral strains in a secondary host
* No cross-protective immunity to virus expressing novel hemagglutinin/neuraminidase
167
What are the features of primary immunodeficiencies?
* Genetic basis
* Majority are diagnosed in children
* Milder forms may not be recognized until adulthood
* About 1 in 500 people is born with a primary immunodeficiency
168
What is the genetic basis of primary immunodeficiencies?
* Inherited
* Familial
* Often monogenic
169
What is monogenic?
Often caused by a single gene mutation or deficiency
170
What are the features of secondary immunodeficiencies?
• Also known as acquired or immunosuppression
• Defect following exposure to
o Disease/infection
o Drug treatment
o Environmental exposure
• Can also be caused by aging and malnutrition
171
How is primary immunodeficiency varied?
* Ethnic origins
| * Phenotype between ethnic origins
172
How does ethnic origins effect primary immunodeficiency disease prevalence?
Prevalence in 100,000 inhabitants in the Middle East and North Africa region compared with countries from other world region
173
What is variation in ethnic origins due to?
* Different ethnicities
| * Familial practices
174
How is chemotaxis of leukocytes achieved?
Leukocyte needs to roll along the surface of the vasculature under the influence of selectins and mucins. Chemokines and chemokine receptors are presented by glycosaminoglycans which activates integrins, firm adhesion and extravasation (migration into tissues)
175
What is opsonization?
Enhancing phagocytosis of antigens
176
What is chemotaxis?
* Directional movement
| * Attracting macrophages and neutrophils
177
What is cell lysis?
Rupturing membranes of foreign cells
178
What is the function of phagocytes?
* Engulf and destroy pathogens using a plethora of microbial agents
* Participate in antigen presentation of engulfed proteins
179
What is the function of NK cells?
* Deal with intracellular pathogens (shielded from phagocytes and complement)
* Antiviral, protect from cancer and malignancies
* Induce apoptosis (by perforin and granzymes) and secrete cytolytic granules
180
What are the functions of dendritic cells?
* Sample environment by pinocytosis (“drinking”) or phagocytosis (“eating”)
* Resident in tissues and mature upon activation and travel to local nodes
* In nodes antigen presentation and co-stimulation
181
What are the functions of CD4 T cells?
* TH1 macrophage activating
* TH2 B cell activating
* Treg control inflammation
182
What are the functions of CD8 T cells?
* Cytotoxic response to infected cells
| * Antiviral and anti-cancer response
183
What are the functions of B cells?
* Memory cells in circulation
| * Plasma cells produced in bone marrow produce antibodies
184
What are the functions of IgM?
* Produced early
* Low affinity
* Activate complement
185
What are the functions of IgA?
Secreted across mucosa to prevent re-infection
186
What are the functions of IgE?
Bind to mast cells to clear multicellular parasites
187
What are the functions of IgG?
* Sterilising/neutralising
* Cross placenta from mother to baby
* Signal NK cells and phagocytes
188
What does PID stand for?
Primary immunodeficiency diseases
189
What is PID?
* They are genetic, familial, and heritable
* They impact culture and society – predominant in cultures with consanguineous marriage
* Predisposed to specific infection – teaches us how the immune system works
190
What defects can occur in PID?
* Defects in pattern recognition
* Defects in phagocytes
* Defects in migration
* Defects in complement
* Defects in B cells
* Defects in T cells
191
What is the defect in pattern recognition in patients with PID?
* Toll-like receptors (TLRs) are crucial in recognition of pathogen associated molecular patterns (PAMPs)
* MyD88 is an adaptor required for signalling
* MyD88 deficiency results in severe life-threatening infection with pyogenic bacteria including pneumococci
192
What is pyogenic?
Pus forming
193
What is an example of a defect in phagocytes in patients with PID
* Chronic granulomatous disease and p91 mutation
* Neutrophils fail to produce reactive oxygen species (superoxide – phorbol ester)
* Poor ability to kill
* Aspergillus fumigatus pneumonitis (an e.g.)
194
What is an example of a defect in migration in PID?
* Lack of CD 18 subunit of β2 integrins results in “Leukocyte adhesion deficiency” (LAD) and recurrent bacterial infections
* Only neutrophils are affected
* Other leukocytes can migrate using β1 integrins
195
What is an example of defects in complement in PID?
* Defective terminal membrane attack complex proteins (C7, C9 or factor D) all results in Neisseria gonorrhoeae and N. meningitidis
* Deficiencies in several other components predisposes to development of autoimmune disease
196
What is an example of defects in B cells?
```
• X-linked agammaglobulinemia that more likely affects males results in grossly depressed Ig levels
o Cell mediated immune responses normal
o Pyogenic infections common
• Selective IgA deficiency
o Most common of all PIDs
o Affects circulating and secretory
```
197
What is an example of defects in T cell?
* Often impact on B-cell function
* DI-George syndrome due to mutations in TBX1 transcription factor T cell development is disturbed (malformed T cells – 6% of normal level)
* Hyper-IgM syndrome due to deficiency in transcription factor STAT3
198
What are the features of Hyper-IgM syndrome due to deficiency to STAT3 in defected T cells?
* Decreasing expression of CD40
* Absence of IgG, IgA and IgE
* Infections with Toxoplasma
199
What does SCID stand for?
Severe combined immunodeficiencies
200
What are the features of SCID?
* Absolute failure in T cell development
* Affects 1 in 8000
* Profound defects in cellular and humoral immunity
* Death within first year of life due to severe and recurrent opportunistic infections
* Vulnerable to vaccination with attenuated organisms (e.g. rota virus vaccine)
201
What are some causes of SCID?
* T-B-NK+ SCID – RAG-1, Rag-2
* T-B-NK- SCID - ADA
* T-B+NK+ SCID – IL-7Rα chain
* T-B+NK- SCID – gamma-c chain – cytokine signalling pathways (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21)
* RD – AK2
* Artemis
* JAK-3
202
What are the causes of secondary immunodeficiencies?
```
• Infection
o HIV
o Measles – increased IL-12 production by virus protein cross-linking CD46
• Side-effects
o X-rays
o Cytotoxic drugs
o Malignancy
```
203
What tests are used to identify immunodeficiencies?
* ELISA – be careful of children (immunoglobulins aren’t all present at birth)
* Test humoral response
* Phenotype lymphocytes by flowcytometry
* Sheep red blood cell lysis – complement
204
What therapies are available for immunodeficiencies?
* Early intervention with antibiotics, antifungals, and antivirals
* Replace missing components
* Gene therapy
* Transplantation
205
What are some examples of replacing missing components in therapies for immunodeficiencies?
* Adenosine deaminase deficiency due to SCID – enzyme can be injected weekly
* Defects in humoral response – pooled Ig from several donors will contain antibodies to many common infections
206
How is gene therapy done?
Retroviral gene therapy vector into a packaging line inserted into the host cell
207
Is gene therapy a success?
* There are some successes and some failures
| * In 2008 in a study for SCID half of the patients developed malignancies and one of them died
208
What are some features of transplantation as a therapy for immunodeficiencies?
* Replace entire immune system with stem cells from healthy donor
* MHC polymorphism is limiting factor – risk of graft-vs-host disease
* Radioblation or cytotoxic drugs may be required to minimise host-vs-graft disease
209
What are the features of haematopoietic stem cell transplantation?
* Conditioning regime of chemo/radio therapy
* Infused with donor haematopoietic stem cells
* Immunodeficiency improves as their immune system is replaced by donor lymphoid cells
210
What are the features of graft vs host disease (GvHD)?
* Characteristic targets of liver, skin, and gut
* Graded according to severity from 0-IV
* Incidence of stage II-IV is 35-50% (5500/year)
* T cell mediated allo-immune response
211
What is the criteria for a GvHD?
* The donor graft contains immune competent cells
* There is an immunological disparity between donor and host
* The host immune system is incapable of mounting a response
* Donor effector cells must be able to migrate to the host target tissue
212
What is the three phases model of GvHD?
* Commonly used to describe the pathophysiology of acute GvHD
* Phase 1 occurs prior to transplantation, the conditioning regime of chemo and/or radio therapy causes damage to the target organs which leads to the production of pro-inflammatory cytokines or ‘danger signals’
* Phase 2 occurs after transplantation, host antigen presenting cells activate donor T cells leading to activation and proliferation of donor T cells.
* Phase 3 is the effector phase where the donor T cells migrate to the host tissues and cause damage by a variety of mechanisms
213
Why in phase 2 of GvHD does activation and proliferation of donor T cells occur?
Because donor T cells are presented with antigens which appear foreign to them due to the genetic differences between the donor and the recipient
214
What is the current clinical management of GvHD?
* Immune suppression
* Treatment with steroids
* Regulatory T cells have been shown to prevent GvHD in mouse models and more recently clinical trials
215
What is the difference between GvHD and HvGD?
* In GvHD mature T cells from graft recognise host cell as foreign
* In HvGD mature T cells in host recognise graft cells as foreign
216
What is the hallmark of a successful graft for immunodeficiency?
No immune response by T cell depleted graft. Stem cells proliferate and reconstitute host immune system
217
What is the demographic of the HIV pandemic?
* Killed 25 million by 2008
* More than 35 million infected
* 3 million new cases in 2008 alone
* 14 million children orphaned
* Females over 16 account for >50% of cases
* Young people (15-24) account for 34% of cases
* First reported case in 1981
* Syndrome characterised with predisposition
* HIV-1 isolated in 1983
218
What are the predispositions of HIV?
* Opportunistic infections
* Aggressive form of Kaposi’s sarcoma
* Concurrent depletion of CD4 cells
219
What are the categories of HIV?
```
• HIV-1
o M
o O
o N
o P
• HIV2
o Eight groups
o Less virulent
```
220
Where is the prevalence for HIV?
* HIV-1M is spread worldwide (subcategories A-K)
| * HIV1-O/N/P and HIV-2 is predominant in West Africa
221
What is the clinical course of HIV?
* Infection
* Acute viremia
* Latency
* Early AIDS
* Terminal AIDS
222
What can infect someone with HIV?
* Exposure to bodily fluids
* Sexual intercourse (most common)
* Intravenous drug delivery (sharing of needles)
* Blood/blood products
* Breastfeeding (25% perinatal transmission)
223
What bodily fluids can infect someone with HIV?
* Semen
* Vaginal fluid
* Milk
* Blood
224
How is someone infected with HIV?
* Intraepithelial dendritic cells bind HIV using DC-SIGN
* HIV is internalized into early endosomes
* Dendritic cells that have migrated to lymph nodes transfer HIV to CD4 T cells
225
What occurs in acute viremia of HIV?
* 2-8 weeks after infection, explosion of viral replication (particularly in the gut)
* Symptoms occur
* Spontaneously resolves after 1-4 weeks
226
What are the symptoms during acute viremia of HIV?
* High fever
* Sore throat
* Headaches
* Swollen lymph nodes
* Diarrhoea
227
How is HIV spontaneously resolved in acute viremia?
* Strong HIV specific CD8 killing of infected cells
| * Production of HIV-specific antibodies
228
What occurs in latency of HIV?
* CD4 levels rebound but still low (800/ml compared with 1200/ml in healthy)
* Baseline level of virus in the blood – a “set point” which is best indicator of individual prognosis
* Gradual decline in number and function of CD4 cells
* Asymptomatic for 2-15 years
229
How does the number and function of CD4 cells decline in latency of HIV?
* Direct cytopathic effect of viral proteins on host cell
* Infected cells have increased susceptibility to apoptosis
* Elimination of infected CD4 cells by CD8 cells
230
What occurs in early AIDS within the clinical course of HIV?
* Number of functional CD4 drops below 400cells/ml
* A drop below 200cells/ml is classified as AIDS
* Opportunistic infections appear to exploit weakened immune system (e.g. early Candida (oral thrush) and M. tuberculosis)
231
What does AIDS stand for?
Acquired immunodeficiency syndrome
232
What occurs in terminal AIDS within the clinical course of HIV?
* Reactivation of latent infections
* Fungal pneumonia – highly fatal without treatment
* Mycobacterium avium and cytomegalovirus – treatment resistant environmental infections
* Respiratory infections are the major cause of death
233
What latent infections can be reactivated in terminal AIDS?
* Shingles (chicken pox)
* B cell lymphomas (Glandular fever, EBV)
* Kaposi’s sarcoma (Herpes)
234
What is the structure of HIV?
* Retrovirus – RNA genome but replication passes through DNA with involvement of reverse transcriptase
* Lentivirus – prolonged course of disease
235
How many genes does the HIV genome have?
10
236
What are the genes of HIV genome?
* LTR (long terminal repeat)
* gag (Pr55gag)
* pol (polymerase)
* vif (viral infectivity factor (p23))
* vpr (viral protein R (p15))
* rev (regulator of viral gene expression (p14))
* tat (transcriptional activator (p14))
* vpu (viral protein U)
* env (gp160 envelope protein)
* nef (negative effector (p24))
237
How does HIV enter the cells?
* Envelope spike which engages CD4 on – T lymphocytes, macrophages and dendritic cells
* Binding event changes shape of gp120 to form a binding site for coreceptors CCR5 and CXCR4
* Leads to gp41 (highly hydrophobic) acting like a harpoon to destabilise T cell membrane and promote fusion of virus to cell
238
How is HIV integrated?
* Genome consists of 2 single stranded RNA molecules
* Reverse transcriptase has no proof reading – 1 mutation per genome per reverse transcription
* Converts to DNA in cytoplasm then translocates to nucleus and preferentially targets areas of active transcription (but can integrate in latent areas)
* Viral sequence now part of genome host cell
239
How is HIV replicated?
• Production of viral transcripts by RNA polymerases and transcription factors of host cell
o Activation of T vells results in expression of NFkB transcription factor, this also binds HIV promotor
• Viral proteins expressed in sequence as needed by alternative splicing of transcripts
• New viron assembled at plasma membrane
240
How is HIV tested for?
* 3rd generation tests
* 4th generation tests
* PCR testing
241
What are the features of 3rd generation tests of HIV?
* Tests for anti-HIV antibodies
* Good for long term screening
* Can give false negatives during window period of initial infection (3 weeks – 2 months, prior to seroconversion, production of anti-HIV antibodies)
242
What are the features of 4th generation tests of HIV?
* Tests for anti-HIV antibodies
* P24 antigen (rises sharply during primary infection)
* Shorter window period
* 20 samples are pooled and then singly tested if any are positive
243
What are the features of PCR testing of HIV?
* Common in USA as alternative to 4th generation (window period 5 days shorter)
* Pools of 90 together, then batches of 10 then singly to identify patients
* Test more expensive
244
What are some potential drug targets in the virus life cycle from most potential to least?
* Reverse transcriptase (current clinical use)
* Entry (CCU)
* Fusion (CCU)
* Integration 9CCU)
* Protein production (CCU)
* Assembly
* Maturation
245
What are the features of HIV therapeutics?
Potential drug targets in the virus life cycle
246
What potential drug targets are in HIV therapeutics?
* Entry, maraviroc (oral, blocks co-receptor CCR5)
* Fusion, enfuviritide (rescue drug, twice daily sub cut, expensive, binds gp41)
* Reverse transcription, nucleotide analogues or allosteric inhibitors
* Integration, raltegravir (oral twice daily)
* Protein production, saquinavir (oral)
247
What does each person vary with drug efficacy?
* Error prone nature of virus
* Large viral load
* Rapid rate of replication
248
What is the drug efficacy and resistance in HIV drugs?
* Each person has many variants
* Some protease inhibitors rendered ineffective by single mutation
* Others (e.g. zidouvudine) require multiple mutations
* Most effective to give a “cocktail” of drugs with different mechanisms
249
What does a HIV vaccine need to be?
* Effective – must evoke protective levels of immunity
* Available
* Stable – preferably not requiring refrigeration
* Cheap
* Safe – eliminate pathogenicity
250
What does current research on HIV aim to generate?
* “blunting” antibodies to protect against disease rather than infection
* Strategies to increase cellular immune responses and reduce viral set point
251
What are the four main cancers in the UK?
* Breast
* Lung
* Large bowel
* Prostate
252
What rank is cancer in the cause of mortality in the UK?
2nd
253
What is cancer caused by?
Progressive growth of the progeny of a single transformed cell
254
What is the simplified process of tumour cell growth?
* Initial modified tumour cell
* The modified tumour cell begins to proliferate into a localised benign tumour
* The tumour cells become invasive into the tissues
* The tumour cells invade blood vessels allowing metastasis to occur
255
What is metastasis?
Spread of cancer to different parts of the body travelled through the blood or lymph
256
What does curing cancer require?
Removal or destruction of all malignant cells without harming the patient
257
What is the leading cancer with the most cases in men?
Prostate
258
What is the leading cancer with the most cases in women?
Breast
259
What is the leading cancer with the most deaths in men and women?
Lung and bronchus
260
Which cancer has the highest survival rate?
Prostate - 99%
261
Which cancer has the second highest survival rate?
Melanoma of the skin - 91%
262
Which cancer has the lowest survival rate?
Pancreas - 4%
263
What can be the causative agents of cancer?
* Chemical carcinogens
* Spontaneous
* UV and ionizing radiation
* Virus-induced
* Genetic abnormalities
* Immunosuppression
264
What are some examples of chemical carcinogens that cause cancer?
* Formaldehyde
* Ethanol
* Methanol (in antifreeze)
265
What are some examples of spontaneous events that cause cancer?
* Random mutation event
* Cell division
* Cell proliferation
266
What are some examples of UV and ionising radiation that cause cancer?
* X-rays
| * Sun exposure
267
What are some examples of virus-induced cancer causing agents?
* Hepatitis C
* Epstein Barr Virus
* Human papillomavirus
268
What are some examples of genetic abnormalities causing cancer?
* Xeroderma pigmentosum (XP)
| * Families can develop errors in DNA proofreading
269
What is xeroderma pigmentosum (XP)?
Decreased ability to repair DNA damage such as that caused by UV light
270
What is the characteristic of normal cells regarding cell renewal and cell death?
* Mature cells have a given life span
| * Replaced by proliferation and differentiation of stem cells
271
What disrupts normal healthy cells to make cancerous cells?
* Chemical
* Physical
* Viral agents
* All that mutate the DNA
272
What are the types of oncogenes?
* v-onv (virus oncogenes)
| * c-onc (cellular oncogenes)
273
What are oncogenes?
A gene that has the potential to cause cancer
274
What type of gene is affected by mutations that induce tumour formation?
* Induce cellular proliferation
* Inhibit cellular proliferation
* Regulate programmed cell death
275
What gene induces cellular proliferation?
Proto-oncogenes
276
What gene inhibits cellular proliferation?
Tumour suppressor genes
277
What gene regulates programmed cell death?
Tumour suppressor genes
278
Why can cells not proliferate indefinitely?
Limited availability of growth factors
279
What is a feature of benign tumours?
* Do not grow indefinitely
| * Do not significantly invade surrounding tissue
280
What is a feature of malignant tumours?
* Become progressively invasive
| * Can metastasise
281
Where are carcinomas derived from?
Endodermal and ectodermal tissues
282
Where are leukaemia and lymphomas derived from?
Hematopoietic cells
283
Where are sarcomas derived from?
Mesodermal connective tissue
284
What is the development of a normal cell to a metastatic cell?
* A multi-step process of clonal evolution driven by several mutations
* It is a sequential discreet evolution of cancer
285
What is the flow order of the development of a normal cell to a metastatic cell?
* Normal
* Hyperplastic
* Dysplastic
* Neoplastic
* Metastatic
286
How are cancer cells different?
* Clonal in origin
* Deregulated growth and lifespan
* Altered tissue affinity
* Resistance to control via apoptotic signals
* Change in surface phenotype and markers
* Structural and biochemical changes
* Presence of tumour-specific antigens
287
What differences of cancer cells to normal cells are responsible for an immune attack?
* Change in surface phenotype and markers
* Structural and biochemical changes
* Presence of tumour-specific antigens
288
When was immune surveillance of cancer proposed and refined?
* Proposed in 1909
| * Refined in 1950s
289
Who proposed and refined immune surveillance of cancer?
* Proposed by Paul Ehrlich
| * Refined by Burnet and Thomas
290
What was the proposed immune surveillance of cancer?
* “In animals … genetic changes must be common and a proportion … will represent a step towards malignancy.
* … there should be some mechanism for eliminating such potentially dangerous mutant cells ad it is postulated that this mechanism is of immunological character”
291
When was evidence of immune surveillance of cancer found?
2001 by Schreiber et al
292
What did the evidence find about immune surveillance of cancer?
IFN-γ and lymphocytes prevent primary tumour development and shape tumour immunogenicity
293
What does more recent studies on immune surveillance of cancer tell us?
* Identify effector populations
* Knockout models utilised
* Nude mice studies
294
What is the evidence for immune surveillance in mice?
* Animal models showed that pre-treatment of mice with killed tumour material could protect against a subsequent challenge
* This is T cell based
295
How were the mice models used in immune surveillance of cancer?
* Immunized mouse with irradiated tumour cells
* In one mouse inject viable cells of same tumour
* In another inject viable cells of a different tumour
* Those with same tumour cells responded with tumour rejection antigens eliminating the tumour
* Those with different tumour cells did not eliminate tumour
296
What is the evidence that immune surveillance of cancer of T based?
* T cell removal from mice removed protection to tumour
| * Transfer of T cells from immunized mouse can protect a naïve mouse from tumour challenge
297
What evidence is there for immune surveillance in humans?
* Immunosuppression leads to increased development of viral-derived tumours
* Organ transplant
* High tumour-infiltrating lymphocyte number correlates with improved survival
* NK cell loss corelates with increased tumour pathogenicity
298
What is an example of viral-derived tumour from immunosuppression?
Kaposi sarcoma from herpes in AIDS
299
How are organ transplants evidence for immune surveillance?
* Increases risk of malignant melanoma
| * 3-fold higher risk of sarcoma
300
What antigens does a cancer cell have?
* Viral
* Silent
* Mutant
* Immunological escape changes
* Loss of organ-specific antigen
* Altered carbohydrate (cell division)
* Metastatic molecules
301
What part of the immune system do tumour antigens trigger?
* Humoral
| * Cell-mediated
302
What tumour antigens are recognised by T cells?
* Antigens only expressed by tumours
* Mutated normal antigens
* Antigens expressed at wrong stage of cell growth
* Overexpressed antigens
303
How does a mutated antigen lead to a new epitope?
* Point mutation in either self-protein or self-peptide
* Mutation in shape (self-peptide) – immune system recognises it as a new antigen
* New epitope within cell (self-protein) due to binding of a new peptide to MHC molecule
304
How does an abnormal expression lead to a new epitope?
* In a normal cell either too little expressed or overexpressed
* Too little results in presentation of antigens novel to the adult immune system
* Overexpressed leads to more of one peptide being presented, allowing recognition by T cell
305
Why is T cell recognition not likely in a solid organ cancer?
* Normally naïve T cells do not move into tissues – do not see tumour antigens
* Tumour does not express co-receptors to activate naïve T cells
* Tumour metastasis to lymph node is too late
* Tumour mutation could prevent CD8 T cells recognition
306
Which tumour mutations could prevent CD8 T cell recognition?
MHC or TAP downregulation
307
Why can T cells recognise cancer cells in haematopoietic cancers?
* Cancerous blood cells encounter naïve T cells
* Some haematopoietic cells express high levels of B7
* Not 100% effective
308
How does the adaptive immune response target tumours?
• When tumour cell is dying, antigens are released
• Antigens are engulfed by APCs taken to lymph node
• Presented to T cell which stimulate:
o Antibody/ADCC/complement is activated by helper T cells that educate other T/B cells what is occurring
o CTL recognise and destroy tumour cells
309
What are some effector mechanisms against cancer that the body releases?
* Monocyte/macrophage release lytic enzymes and phagocytose necrotic cells
* Antibody against tumour antigens
* Initiation of NK cytotoxic responses
* Induction of tumour specific CD8 T cells
* Promotes proinflammatory anti-cancer environment
310
What responses are initiated by NK cells as an effector mechanism against cancer?
* Antibody-dependant cellular cytotoxicity (ADCC)
| * MHC I surveillance
311
What does induction of tumour specific CD8 T cells do as an effector mechanism?
CD8 T cell migrates into tumour site and initiates cytotoxic responses
312
How does the effector mechanism of the body promote pro-inflammatory anti-cancer environment?
* Release cytokines/chemokines (TNFα, IFNs)
| * Release antiangiogenic factors
313
How are tumour cells killed by NK or CD8 T cells?
1) Class I + Ag on the tumour cell binds to TCR on the CTL
This triggers perforin and granzyme B to be released
Perforates tumour cell
2) Fas (CD95) on the tumour cell bind to FasL on the CTL
This triggers tumour cell death
314
What is the immune response tumour elimination?
* Innate immune response recognises tumour cell establishment
* NK cells and other effectors recruited to site by chemokines – target tumour growth directly
* Tumour-specific T cells home to site along with macrophages and other effectors to eliminate tumour cells
315
When does immune response tumour elimination occur?
Early in tumour development
316
What is a drawback of immune response tumour elimination?
Not all tumour cells will be killed
317
What is one way that 'clever' cancer cells use to escape attack?
Downregulate MHC I to evade T cells
318
What is the problem for cancer cells with downregulated MHC I on a cancer cell?
* NK cells desire to kill but this is negated by high expression of MHC I on our healthy cells
* So, the ‘clever’ cancer cells with low expression of MHC I will be killed by NK cells
319
What is evidence of NK cell control of cancer?
Maintenance of remission in acute leukaemia's dependent upon CD56+/CD8α+ NK cells – Lowdell et al (2002)
320
What ways can tumour cells evade the immune response?
* Downregulation of MHC I and TAP
| * May provide poor co-stimulation
321
What are the three E's of immunoediting?
* Elimination
* Equilibrium
* Escape
322
What occurs in elimination of immunoediting?
* NK, CTL and CD4 cells kill tumour cells
| * Not all
323
What occurs in equilibrium of immunoediting?
There is the same number of immune cells to tumour cells
324
What occurs in escape of immunoediting?
Cells avoid immunological control and proliferate
325
What mechanisms by which tumours can avoid immune recognition?
* Low immunogenicity
* Tumour treated as self-antigen
* Antigenic modulation
* Tumour-induced immune suppression
* Tumour-induced privileged site
326
What does low immunogenicity involve?
* No peptide: MHC ligand
* No adhesion molecules
* No co-stimulatory molecules
* No way of cells binding to the tumour
327
What does antigenic modulation involve?
* Antibody against tumour cell-surface antigens induces endocytosis and degradation of antigen. Immune selection of antigen-loss variants
* Antibody binds to the surface of the tumour which engulfs, and lysis occurs
328
What does tumour treated as self-antigen involve?
* Tumour antigens taken up and presented by APCs in absence of co-stimulation tolerize T cells
* This induces tolerance in the form of anergy (prolonged signal 1, no signal 2)
329
What does tumour-induced immune suppression involve?
Factors secreted by tumour cells inhibit T cell and induces Treg
330
What does tumour-induced privileged site involve?
Factors secreted by tumour cells create a physical barrier to the immune system
331
What factors are secreted in tumour-induced immune suppression?
* TGFβ
| * IL-10
332
What does TGFβ do?
* Important in the function of regulatory T cells
| * Actively inhibits T cell proliferation
333
What does IL-10 do?
Actively inhibits maturation of dendritic cells
334
What are the features of an immature dendritic cell?
* Static – keeps position in tissues
* Constantly phagocytosing
* Low surface class II MHC
* No expression of co-stimulatory molecules
335
What are the features of a mature dendritic cell?
* Highly mobile – migrates to lymph
* Stops phagocytosing
* High surface class II MHC
* High expression of co-stimulatory molecules
336
How does an immune tolerance generate with dendritic cells?
* Signal 1 (antigen) is present
* Signal 2 (co-stimulation – danger signal) – not present
* Dendritic cell not activated
* Tumour cells in lymph are perceived not dangerous
* Anergy (peripheral tolerance)
337
What are the types of tumour associated macrophages?
* M1
| * M2
338
What is M1 activated in the presence of?
IFN-γ
339
What is M2 activated in the presence of?
IL-10
340
What are the features of M1?
* Defence against bacteria
* Tumour suppression
* Immune stimulation
341
What are the features of M2?
* Tissue repair and angiogenesis
* Tumour promotion
* Downregulation of M1 and adaptive immunity
342
What are the types of anti-cancer therapies?
* Radiation
* Alkylating
* Anti-metabolites
* Natural products
* Metals
* New drugs
343
What forms can immunotherapy take?
* Passive transfer
| * Active immunization/vaccination/immunotherapy
344
What is passive transfer in immunotherapy?
Anti-tumour antibody (a range of monoclonal antibodies)
345
What is active immunization in immunotherapy?
Strategies that generate an anti-tumour immune response (normally cytotoxic T cells)
346
What are tumour rejection antigens?
* The antigens expressed by experimentally induced murine tumours
* Peptides of tumour cell proteins that are presented to T ells by MCH molecules
347
What are the types of antigens that are targets of T cell responses in tumour rejection antigens?
* Antigens not normally expressed on somatic cells
| * Antigens that are normally expressed on somatic cells
348
What are some tumour-specific antigens?
* Bcr-abl
| * CDK-4/β-catenin
349
What cancer is Bcr-abl associated with?
Chronic myeloid leukaemia
350
What cancer is CDK-4/β-catenin associated with?
Melanoma
351
What are some testes-specific antigens?
* MAGE 1-3
| * NY-ESO-1
352
What cancer is MAGE 1-3 associated with?
* Melanoma
| * Colorectal cancers
353
What cancer is NY-ESO-1 associated with?
Melanoma
354
What are some differentiation antigens (found in the womb in development)?
* Tyrosinase (TRP-1/2)
* Melan-A
* Monoclonal Ab
355
What cancer is Tyrosine (TRP-1/2) associated with?
Melanoma
356
What cancer is Melan-A associated with?
Melanoma
357
What cancer is Monoclonal Ab associated with?
Myeloma
358
What are some tumour-associated antigens?
* MUC-1
* α-fetoprotein
* Her-2/neu
* WT-1
* Myeloblastin
* Survinin
359
What cancer is MUC-1 associated with?
* Myeloma
* Breast
* etc
360
What cancer is α-fetoprotein associated with?
* Liver
* Ovaries
* Testes
* Lymphoma
* etc
361
What cancer is HER-2/neu associated with?
Breast
362
What cancer is WT-1 associated with?
* Many
* Leukaemia
* Solid tumours
363
What cancer is myeloblastin associated with?
Leukaemia
364
What cancer is Survinin associated with?
Many
365
What are some antigens that are not normally expressed on somatic cells?
* Antigens only expressed in embryo
* Gene products of mutated or recombined cellular genes
* Sequestered antigens (cancer-testis family – apart from tumours only expressed in testis)
* Viral antigens
366
In principle what is the ideal target for immunotherapy?
* Immune response not limited by self-tolerance
| * Responses to the antigens are unlikely to inflict damage on normal healthy cells
367
What percentage of colorectal cancers have MAGE-3 expressed?
25%
368
What can vaccines containing MAGE-3 induce?
* Both tumour regression and cytotoxic T cell response in patients with advanced melanoma
* Tumour regression correlates with the number of anti-MAGE-3 CD8 T cells
369
What cancer does human papillomavirus (HPV) cause?
Cervical cancer
370
What percentage of American women have contracted at least one strain of HPV by the age of 50?
More than 80%
371
What rank is cervical cancer on the deadliest cancers of women?
5th
372
How many HPV strains are there?
120
373
How many HPV strains cause cervical cancer
20
374
Which strains of HPV are the more dominant types in cervical cancer?
* HPV 16
| * HPV 18
375
Which oncogenes are responsible for cervical carcinogenesis?
* E7
| * E6
376
What does E7 do in cervical cancer?
E7 causes uncontrolled cell proliferation as it complexes with Rb gene
377
What does E7 do in cervical cancer?
E6 causes the loss of DNA repair function and prevents cell undergoing apoptosis as it binds to and degrades p53 gene protein product
378
What HPV strains does Gardasil protect against?
* HPV 6
* HPV 11
* HPV 16
* HPV 18
379
What vaccine is used for HPV cervical cancer?
DNA free virus like particles (VLP) synthesised by self-assembly of the fusion proteins of the major capsid antigen L1 induce strong humoral response with neutralizing antibodies
380
What are the trade names for HPV vaccine for cervical cancer?
* Gardasil
| * Cervarix
381
What trials were done for HPV vaccine in cervical cancer?
MERCK and GSK using HPV 16 and HPV 18 L1
382
What are some potential tumour antigen targets?
* Tissue specific antigens – melanoma/melanocyte antigens
| * Antigens that are widely expressed at very low levels on normal tissues
383
What must be considered when using tissue specific antigens and low level widely expressed antigens?
* Existence of immunological tolerance
| * Possibility of inducing autoimmune diseases
384
What are some anti-cancer antibody immunotherapies?
* Naked Mab
* Immunoconjugates
* Multistep targeting
385
What does naked Mab trigger?
* Antibody-dependent cell-mediated cytotoxicity (ADCC)
| * Complement-dependent cytotoxicity (CDC)
386
What are some immunoconjugates?
* Radioimmunoconjugate (radionuclide binds to antibody)
* Immunocytokine (cytokine binds to antibody)
* Immunotoxin (toxin binds to antibody)
* Antibody-directed enzyme prodrug therapy (ADEPT)
* Immunoliposome
* Cellular immunoconjugates (killer cells binding to antibody)
387
What is multistep targeting?
* Biotinylated radioactive ligand binds to streptavidin (amplifier) which is bound to an antigen on the surface of the tumour cell
* Biospecific MAb (monoclonal antibody) on antibody binds to radionuclide
388
What does Rituximab (Rituxan) target?
CD20 cells in Non-Hodgkins Lymphoma found on cancerous B cells
389
When was rituximab approed?
1997
390
What type of antibody is rituximab?
Chimeric IgG1
391
What is a drawback of rituximab?
Kills all B cells - even normal healthy B cells
392
What is rituximab used in?
* Autoimmunity
| * Transplantation
393
What does rituximab do?
Destroys CD20 cells found on cancerous B cells either by antibody mediated cellular cytotoxicity or complement dependent cytotoxicity
394
How does complement dependent cytotoxicity kill the B cell?
* C3 -> C3b
* C3b binds to MAC (membrane attack complex) or antibody bound to CD20
* This activates cell death
395
How does antibody-dependent cell-mediated cytotoxicity kill the B cell?
* FcγR/CR-mediated opsonic phagocytosis – FcγR binds to antibody bound to CD20 – activates phagocytosis cell death
* Complement receptor on macrophage binds to rituximab bound to antibody bound to CD20 on B cell – activates cell death
396
How does NK cells kill the B cell?
Same as macrophage killing but with a NK cell ending in direct lysis with perforin and granzyme B
397
What does Trastuzumab (Herceptin) target?
Targets HER-2 (human epidermal growth factor receptor 2) which is expressed at high levels in some breast cancers
398
What antibody is used in Trastuzumab?
Humanized IgG1
399
When was Trastuzumab approved?
1998
400
What does Trastuzumab do?
* Acts by the inhibition of HER-2 mediated tumour cell proliferation and migration
* It is only active in cells that overexpress HER-2
401
What percentage of breast cancer patients overexpress HER-2?
25%
402
What does Gemtuzumab ozagamicin (mylotrag) target?
Targets CD33 which is expressed in 80% acute myeloid leukaemia cases but not in normal hematopoietic cells
403
What antibody is used in Gemtuzumab ozagamicin?
Humanized IgG4
404
What is humanized IgG4 linked/bound to in Gemtuzumab ozagamicin?
Calicheamicin
405
When was Gemtuzumab ozagamicin approved?
2000
406
What dpes Gemtuzumab ozagamicin do?
* Antibody binds to CD33 resulting in a complex which is internalised
* The calicheamicin derivative is released into the lysosome of the myeloid cell
* The calicheamicin derivative binds to DNA resulting in the breakage of the strands and apoptosis
407
What does Alemtuzumab (Campath) target?
Targets CD52 which is expressed on B cells in patients with B cell chronic lymphocytic lymphoma
408
What antibody is used in Alemtuzumab?
Humanized IgG1
409
When was Alemtuzumab approved?
2001
410
What does Alemtuzumab do?
Acts by antibody-dependant cellular cytotoxicity and complement dependent cytotoxicity
411
What is Alemtuzumab used in?
* Transplants
| * Under trail for MS (multiple sclerosis)
412
What does Ibritumomab tiuxetetan (Zevalin) target?
CD20 on B cells in Non-Hodgkins Lymphoma
413
What antibody is used in Ibritumomab tiuxetetan?
Murine IgG1 90Y conjugate
414
When was Ibritumomab tiuxetetan approved?
2002
415
What does Ibritumomab tiuxetetan do?
* Delivery of cytotoxic radiation leading to apoptosis
* Yttrium 90 decays by emission of Beta particles with a half-life of 64 hours
* Also induces ADCC and CDC
416
What is Ipilimumab used for?
Melanoma treatement
417
What antibody is used in Ipilimumab (Yervoy)?
Human monoclonal antibody
418
What treatment is Ipilimumab undergoing clinical trials for?
* Non-small cell lung carcinoma (NSCLC)
* Small cell lung cancer (SCLC)
* Metastatic hormone-refractory prostate cancer
419
What does Ipilimumab do?
* Blocks CTLA-4 interaction with B7, preventing anti-inflammatory signalling
* CTLA-4 is on the T cell and B7 on tumour cell
Clinical Immunology
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