CMB2004 Cell and Molecular Biology of the Immune System Flashcards
What is specific immunity mediated by?
B and T lymphocytes
What are BCR expressed by?
B lymphocytes
What are TCR expressed by?
T lymphocytes
Which are found only in the membrane form, BCR or TCR?
TCR
What do BCRs bind?
“Free” antigens
What do TCRs bind?
Peptide fragment (epitope) bound to MHC expressed by APC
Describe the structure of antibodies
-Paired Variable (V) regions form two identical antigen binding site
-Constant regions are responsible for antibody structure and interacting with other molecules (FcR) and cells of innate system.
What are antibodies responsible for?
-Agglutination (against pathogens)
-Activating complement
-Activating effector cells
What are the two types of Light chain in antibodies?
Lambda or Kappa
What are the 5 isotypes of antibodies?
IgM, IgD, IgA, IgG and IgE
How many domains are found in the Light chain of an antibody?
2
How many domains are found in the Heavy chain of an antibody?
4 or 5
What comprises each domain in an antibody?
~110 amino acids
-Two Beta sheets
-Linked by a disulphide bridge
-Domains are paired
Name some compounds found in the immunoglobulin superfamily
TCR, MHC class I and II, CD4, CD8
How many hyper variable regions are found in antibodies?
3 in Variable Heavy and Variable light domains
What do the hyper variable regions found in antibodies contribute to?
Complementary determining regions (CDR1-3)
What types of epitopes are there?
Continuous or conformational
Describe the structure of TCRs
-Heterodimer of ⍺ or β chain (sometimes γ or 𝛿)
-Each chain has a V and a C region
-Domains are Ig like
Which cell types are MHC Class I molecules expressed by?
Nearly all cell types in body
Which cell types are MHC Class II molecules expressed by?
Specialised group of immune cells
-ANTIGEN PRESENTING CELLS
Describe the structure of MHC Class I molecules
Heterodimer of ⍺ chain (consisting of ⍺1, ⍺2, and ⍺3) and β2-microglobulin
What are the three different MHC Class I molecules?
HLA-A, HLA-B and HLA-C
What are the different HLA molecules encoded by?
Separate ⍺ chain genes
What do the ⍺1 and ⍺2 domains of MHC class I fold to form?
β sheet structure known as peptide binding site
Describe the structure of MHC Class II molecules
Heterodimer of ⍺ chain (⍺1 and ⍺2 domains) and β chain (β1 and β2 domains)
What are the three different MHC Class II molecule types?
HLA-DP, HLA-DQ and HLA-DR
What do both the ⍺2 and β2 domains of MHC Class II form?
Ig-like structure
How many gene segments make up the H chain and TCRβ in an TCR or BCR?
3 - V, D and J
How many gene segments make up the L chain and TCR⍺ in an TCR or BCR?
2 - V and J
What occurs to the different gene segments during lymphocyte differentiation?
They will rearrange
Describe the rearrangement of Immunoglobulin genes during B cell development.
-DNA containing the Ig gene segments is deliberately broken and rearranged to form functional genes
-Each individual B cell will perform both the breakage and rearrangement randomly
What is the process whereby Ig Gene segments rearrange
Non-homologous End Joining (NHEJ) recombination
Describe the recombination of gene segments to encode the V region of the light chain of an immunoglobulin
-After DNA breaks, a single V and a single J gene segment are joined together
What are the two loci containing L chain immunoglobulin genes?
κ and 𝞴
In what order do Immunoglobulin rearrangements occur?
-First H chain gene segments rearrange
-Then light chain gene segments rearrange forming κ segments first. If this is unsuccessful then 𝞴 segments rearrange.
Describe the recombination of gene segments to encode the V region of the heavy chain of an immunoglobulin
After DNA breaks, a single random V, D and J segments are randomly joined together
There multiple V, D and J immunoglobulin segments, but where are they encoded?
At 3 different loci
-H (on chromosome 14)
-κ (on chromosome 2)
-𝞴 (on chromosome 22)
What is Immunoglobulin gene segment rearranged guided by?
Recombination signal sequences
What complex is involved in the immunoglobulin gene segment rearrangement?
V(D)J recombinase
What genes activate the recombination process in immunoglobulin gene rearrangement?
RAG1 and RAG2 (recombination activating gene)
What occurs if mutations occur in RAG genes?
Immunodeficiency
What is immunoglobulin light chain isotype exclusion
-Each B cell expresses either a rearranged κ or 𝞴 light chain but never both
-Ensures that each individual B cell produces just one randomly generated BCR that is different from every other BCR
Give the mechanisms for generation of antibody diversity
1) Multiple gene segments for each chain
2) Combinatorial diversity
3) Different combinations of heavy and light chains
4) Junctional diversity
5) Somatic hypermutation
Describe how junctional diversity increases immunoglobulin diversity
imprecise joining (small differences in sequences where V-D and D-J segments join)
-N regions (random addition of nucleotides at junctions of V-D and D-J by terminal transferase)
Describe how somatic hypermutation increases immunoglobulin diversity
-Mutation frequency in antibody, V genes us orders of magnitude higher than that seen in all other areas of the genome
-SHM occurs in germinal centres
What carries out Somatic hypermutation in immunoglobulin diversity?
-Activation induced deaminase
-Acts on DNA to deaminate cytosine to uracil
-Uracil is Recognised by error prone DNA repair pathways leading to mutations
The constant region of each immunoglobulin heavy chain is encoded by?
-Different C region gene segment (eg C𝛿, Cµ, Cε)
-Four γ chain gene segments correspond to the four IgG subclasses
What is the first isotype of BCR expressed by developing B cells?
IgM
What can IgM be coexpressed with, and why?
C𝛿 is next to Cµ, hence IgD can be coexpressed with IgM, by differential processing of the RNA from the two C region genes
What are the variable regions in T cell receptors encoded by?
V, D and J segments
Where does Gene segment rearrangement occur in the body in TCRs?
The thymus
In what lymphocytes does somatic hypermutation occur?
ONLY in BCR
Does gene rearrangement occur in MHC molecules
NO
What are the subtypes of genes encoding MHC Class II
HLA-DP, HLA-DQ, HLA-DR
If heterozygous at each MHC Class I molecule, how many different Class I molecules can be expressed (and likewise for Class II)?
6
What is the benefit to having a high level of MHC polymorphism?
-Allows a wide range of epitopes to bind
-Increasing variety of APCs
-Population can respond to almost unlimited number of pathogens/antigens
What is the drawback(s) of having a high level of MHC polymorphism?
-Increased risk of immune-mediated disease (eg autoimmune)
-Reduces pool of available donor organs (as reduced matching)
Where are peptides derived from antigens synthesised/processed inside the cell presented?
Usually by Class I MHC molecules
Where are peptides derived from antigens synthesised/processed outside the cell presented?
Usually by Class II MHC molecules
Describe the process by which antigens are processed and presented by MHC Class I molecules
1 - Antigen synthesised in cytoplasm
2 - Protein cleaved to peptides by proteasome
3 - Peptides transported to endoplasmic reticulum by TAP transporter
4 - Peptides bind to MHC Class I molecules
5 - MHC-1/peptide complex then transported to cell surface
What transports antigens cleaved to epitopes into the endoplasmic reticulum?
TAP transporter
How may proteasomes involved in antigen processing change during inflammation?
Proteasomes receiving inflammatory cytokines signals produce altered peptides
Describe the process by which antigens are processed and presented by MHC Class I molecules
1 - Antigens are endocytose into intracellular vesicles inside the cell
2 - Protein cleaved to peptides by acid proteases
3 - Vesicles fuse with vesicles containing MHC class II molecules
4 - Peptides bind MHC Class II molecules
5 - MHCII/peptide complex then transported inside vesicles to cell surface
Describe how MHC Class II molecules prevent inappropriate binding?
-MHC Class II molecules bind to invariant chain in the endoplasmic reticulum
-Preventing peptides binding in the groove
-In endocytic pathway lysosomal enzymes degrade this leaving CLIP peptide associated with the binding groove
-Peptides from antigen displace CLIP when they bind
-HLA-DM is required for loading of peptides into the groove
What is the role of HLA-DM, a molecule encoded in the Class II region of the MHC HLA?
Required for loading of peptides into the groove of MHC Class II molecules
What do MHC I and II molecules bind and present in normal healthy cells?
Peptides from self proteins
Where are each accessory molecules involved in antigen processing and presenting encoded?
They are all encoded in the MHC
What will happen to any cell expressing MHC Class I molecules with a non-self antigen?
They will be recognised and killed by cytotoxic CD8+ T cells
What will happen to any cell expressing MHC Class II molecules with a non-self antigen?
They will be recognised and activated helper CD4+ T cells
What do B cells develop from?
-Haematopoietic stem cells in bone marrow
-Which express PAX5 transcription factor
What B cell specific markers are expressed?
CD45
What is expressed after B cells are matured?
CD19
Describe how pre B cells mature
-H chain genes rearrange first (µ chain), which then moves to cell surface with Ig⍺ and Igβ and expressed with surrogate light chain
-Then light chains rearrange, displacing the surrogate light chain
What makes up the surrogate light chain expressed in pre-B cells?
VpreB and 𝞴5 chains
Once pre-BCRs are functional, what does the signal sent to preB cells do?
-Turns off RAG1 and RAG2
-5 or 6 rounds of cell division
-Surrogate light chain expression stops
-RAG1 and RAG2 turned on again for gene rearrangement
-L chain rearrangement starts
What occurs if cell fails to productively rearrange both H and L genes?
It dies
Following successful H chain rearrangement, what happens if pre-B cells initially fail to generate non-productive rearrangements of light chain κ genes?
-They can be rescued by up to 10 further rearrangements at the same locus
-If after these attempts and still out of frame, then 𝞴 locus will begin to rearrange
What happens to immature B cells that bind multivalent self antigens?
-Clonal deletion (cell apoptosises)
OR
-Receptor editing (further light chain gene rearrangements of variable regions)
What happens to immature B cells that bind soluble self antigens?
Cell becomes unresponsive (anergic)
Where do premature T cells rearrange receptor genes?
In the thymus
How is T cell development similar to that of B cells?
-Originate from bone marrow stem cells
-Rearrange receptor genes
-Express pre-T receptor
-Elimination of self reactive T cells by negative selection
T cells expressing ⍺β TCR must do what to be selected?
Bind with self MHC (through positive selection)
What occurs after T cells migrate to the thymus?
Develop into thymocytes by
-Rearranging TCR genes (β first) and express TCR
-Acquire other markers eg CD3, CD4, CD8
-Undergo positive and negative selection
Describe the thymus
-Bi lobed organ in anterior mediastinum
-Each lobe divided into many lobules
-Each lobule has outer cortex and inner medulla
Once inside the thymus, describe how thymocytes rearrange TCR genes
-Firstly they rearrange TCRβ genes
-Expressed along with pre-T cell receptor
-Cells proliferate and then rearrange TCR⍺ genes
What does TCR expression require?
The CD3 complex
-CD3 transmits signal to T cell nucleus following TCR recognition of peptide-MHC
What will some thymocytes express instead of ⍺β TCR?
γ𝛿
T cells expressing a randomly rearranged ⍺/β TCRs may
-Recognise self MHC plus peptide from “foreign” Antigen (immunity) - KEPT
-Recognise self MHC plus peptide from “self” Antigen (autoimmunity) - ELIMINATED
-Not be able to recognise self MHC - ELIMINATED
Describe positive selection of T cells
-Positive selection of cells which recognise MHC+Self peptide
-Occurs when double positive (DP, CD4 CD8) T cells recognise MHC on cortical epithelial cells in thymus
-Apoptosise if not recognised
Describe negative selection of T cells
-Negative selection of T cells which recognise MHC+self peptide on thymic dendritic cells/macrophages with high affinity
-TCR binding to MHC/self peptide with high affinity causes T cell to die apoptosis (clonal deletion)
Where does the positive selection of ⍺:β T cells occur?
In the cortical epithelial cells in the thymus
Where does the negative selection of ⍺:β T cells occur?
In the
-Dendritic cells
-Macrophages
-Other cells in the thymus
How does TCR affinity for self peptide-MHC affect selection?
-All T cells recognising self MHC are positively selected
-Those with the highest affinity for MHC+self peptide are then negatively selected
Describe the T cells that survive thymic selection
-Express TCR capable of binding self MHC
-Are depleted of self reactive cells
-Exit the thymus as mature, single positive T cells
Which T cells recognise antigens in association with MHC Class I?
CD8+ T cells
Which T cells recognise antigens in association with MHC Class II?
CD4+ T cells
Following exit from the thymus, through what do naive T cells recirculate, and where to?
-Via blood/lymphatics through High Endothelial Venules (HEV)
-To secondary lymphoid tissue
What occurs when naive T cells make contact with specific APC-Ag?
Clonal proliferation and differentiation
What may naive T cells differentiate into following contact with specific APC-Ag?
-Effector T cells (CD4 or CD8)
-Memory T cells
Following activation in the secondary lymphoid tissue, where may T cells go?
Effectors will migrate to sites of infection
Apart from presenting antigens, how may APCs activate lymphocytes?
Using cytokines
What occurs to T cells that are not activated?
Leave the lymph node via cortical sinuses into the lymphatics, reentering circulation and being recycled for another day.
How may T cells get to where they need to be?
Chemokine receptors expressed on surface of T cells bind chemokines expressed by other cells
Once T cells are physically close to other cells, what will mediate cell/cell interactions?
Cell adhesion molecules
Describe the process that occurs when T lymphocytes contact APC
-T cells contact APCs using CAMs
-TCR scans APC peptide/MHC complexes
-If no recognition they may disengage, if there is recognition CD3 signal activates
-Increasing affinity of CAM interactions
-T cell divides and progeny differentiate to effector cells
What is LFA-1?
Leukocyte function-associated antigen
What is ICAM-1?
Intercellular adhesion molecule
Describe the process by which T cells bind and become activated on a molecular level.
-T cells initially bind APC through low affinity LFA1:ICAM1 interactions
-Subsequent binding of T cell receptors signals LFA1
-Conformational change in LFA1 increases affinity and prolongs cell cell contact
Name the signals involved in T cell costimulation
Signal 1 - TCR contacting MHC-Epitope on APC, involving CD3
Signal 2 - APC express B7.1/2 that bind CD28 on T cells
Signal 3 - APC release cytokines that bind cytokine receptors on T cells
Once activated by costimulation, describe negative feedback in T cell proliferation
-Activated T cells now proliferate and express ICOS and CTLA4
-Binding of CTLA4 to B7.1/2 on APC delivers a negative signal to the activated T cell
What does ICOS on activated T cells bind to?
ICOS (related to CD28) binds ICOSL on APC to induce cytokine secretion by T cells
What does CTLA-4 on activated T cells bind to?
CTLA-4 is highly related to CD28, and shows stronger binding to B7.1/2 than CD28
What are mutations in CTLA-4 associated with?
Autoimmune diseases eg Type I diabetes
APCs activate T cells, but what activates APCs?
-APCs express receptors for microbial molecules (PRR)
-Binding these pathogen associated molecules activates APC
-Leading to up regulation of MHC and Costimulatory molecules
-Ensuring signal 2 to activate T cell mediated response only occurs during infection
How do different cytokines affect differentiation?
Different cytokines dictate the differentiation of activated CD4 cells into different subsets of effector cells
What transcription factor does TGFβ act upon, and what subtype of CD4 does it cause differentiation into?
-Acts on FoxP3
-Differentiating into Treg cells
What transcription factor does IL6 act upon, and what subtype of CD4 does it cause differentiation into?
-Acts on Bcl6
-Differentiating into T follicular helper cells
What transcription factor does TGFβ-IL6 act upon, and what subtype of CD4 does it cause differentiation into?
-Acts on RORγT
-Differentiating into TH17 cells
What transcription factor does IL12-IFNγ act upon, and what subtype of CD4 does it cause differentiation into?
-Acts on T bet
-Differentiating into TH1 cells
What transcription factor does IL-4 act upon, and what subtype of CD4 does it cause differentiation into?
-Acts on GATA3
-Differentiating into TH2 cells
Give some APCs that express MHC Class II molecules II
-Dendritic cells
-Macrophages
-B cells
Name some types of dendritic cells
-Myeloid (conventional DC2,3)
-Plasmacytoid DC (pDC, DC6)
Describe myeloid Dendritic cells (DC2,3)
-Key APC that initiates T cell responses
-Bone marrow derived
-Do not express B7 until activated/matured
-Induced to mature and migrate to lymph node following “danger signal” activation
How may mature myeloid dendritic cells (DC2,3) differ from inactivated DC?
-Found in T cell areas of lymphoid tissues
-DC MHC Class I and II will be loaded with peptides from pathogens they encountered in peripheral tissues
-Their levels of costimulatory molecules will be very high
-They will express high levels of adhesion molecules
What is special about dendritic cells (DC1)?
-They take up and process exogenous antigens and present it via MHC Class I molecules
-This allows these DCs to activate naive CD8 T cells, meaning these (which don’t need co-stim) can kill infected cells that are not APC
Describe macrophages
-Function as scavengers of pathogens but also important APC for extracellular pathogens
-Highly phagocytic
-Express MHC II and B7 which increases following T cell help
-Resident in many tissues at peripheral sites as well as in lymphoid tissue
-Once activated by T cells secrete many inflammatory cytokines
Describe B lymphocytes as antigen presenting cells
-Very poor at phagocytosis
-Internalise soluble Ag for processing and presentation by BCR (act as Ag specific APC)
-Ag binding to BCR up regulates B7 (can provide signal 2 to activate T cells)
What is Interleukin 2?
-A key cytokine for T cell survival
-Autocrine T cell Growth factor
-Once activated T cells express a high affinity IL2R and secrete IL2
-Leading to lots of T cell proliferation
How may Interleukin 2 receptors change once T lymphocytes are activated?
-Naive T cells express a low affinity form of the IL2R
-Activated T cells express a high affinity IL2R and secrete IL2
What is the target of cyclosporin, an immunosuppressive drug?
Interleukin 2
Describe activation of CD8 T cells
-Requires high levels of costimulator activity
-CD8 T cells can be activated directly by infected or cross presenting APC
-Or may require additional help from CD4 T cells.
How are B lymphocytes and T lymphocytes similar in terms of activation?
Both require costimulation ie multiple signals
What is signal 1 in B lymphocyte activation?
Binding of antigen to B cell receptor
What immunoglobulins are expressed on the surface of B cell receptors?
IgM and IgD
What does crosslinking of BCRs in B lymphocyte activation activate?
Intracellular kinases
What is associated with membrane BCRs?
Ig⍺ and Igβ, which contain Immunoreceptor tyrosine based activation motifs (ITAM)
What can enhance signal 1 in B lymphocyte activation?
-Activated complement cascade
-ie lots of C3b
-binding to complement receptor (CR2) on B cell surface
-Aids in forming the BCR coreceptor complex
-Augmenting the signal
What are the two types of antigens that B cells can bind to?
-Thymus independent antigen (TI)
-Thymus dependent antigen (TD)
How do B cells receive signal 2 when activating by TI antigens?
Signal 2 is provided by the antigen itself or extensive cross linking of BCR
How do B cells receive signal 2 when activating by TD antigens?
Signal 2 is provided by CD4 T cells
Describe thymus independent (TI) antigens
Lead to antibody production (only IgM) with no requirement for T cell involvement
Describe the TI1 isotype of Thymus independent antigens
-Binds to other receptors (as well as BCR) on B cells providing signal 2
-The two signals lead to B cell activation, proliferation and antibody secretion
In high concentrations, what do TI1 thymus independent antigens do?
Act as polyclonal activators (mitogens) for B cells
Describe the TI2 isotype of thymus independent antigens
-Often repeated epitopes (eg polysaccharides)
-Will therefore cross link many BCR molecules on same B cell surface
-Take longer to induce B cell activation
At what age do antibody responses to TI2 thymus independent antigens develop?
After 5 years of age
Describe Thymus dependent antigens?
-Antibodies to these antigens require CD4 T cells
-Antibody responses are much better
Describe how all classes of antibodies can be produced to neutralise Thymus dependent antigens
-T cells activated by MHC-epitope on APC
-BCR binds antigen (signal 1)
-Then B cell internalises antigen, processes and presents Ag to CD4 T cells (signal 2) via CD40-CD40L
-Cytokines secreted by T cell aid B cell to class switch
How may we improve the efficiency of a vaccine against pathogens that have T1 antigens?
By converting a TI antigen to a TD antigen
Describe how we may convert a TI antigen to a TD antigen in a vaccine?
-Using a conjugate vaccine
-Coupling a pathogen to a protein on a TD antigen
-Allows young children to be immunised and protected
Describe the process through which B lymphocytes and CD4 T cell interactions occur in the thymus
-B cells enter lymph node from blood
-If a B cell comes into contact with its specific Ag it can then be activated
-If Ag is TD, B cell presents peptide from Ag to CD4 Th cells at the boundary of the T/B areas forming a B/T cell conjugate
Describe the molecular processes through which B lymphocytes and CD4 T cells interact
-B cell presents Ag-BCR to activated CD4 Th cell
-T cell expresses CD40 ligand, secreting cytokines
-B cell receives signal 2 via CD40/CD40L binding and via cytokine from T cells binding receptors causing proliferation
-CD40 signal also induces activation induced deaminase (AID) which causes class switching and SHM
Where in the thymus do B/CD4 T cell interactions occur?
In the boundary between the B cell follicle and T cell zone
Following conjugation of B lymphoblasts and T cells, what occurs?
-These conjugates move to primary follicles (B cell areas) forming Germinal centres within a B cell follicle in secondary lymphoid tissues
-B cells divide rapidly to become centroblasts and then differentiate into non dividing centrocytes
How do B cells differentiate into centrocytes from centroblasts?
Through - Somatic hypermutation of Ig genes
- Isotype switching
Once in a germinal centre, what do B cells do?
-Differentiate into plasma cells (secreting various isotypes)
-or Form long-lived memory cells and recirculate
-or Die within lymphoid tissue, if BCR no longer binds antigen because of somatic hypermutation
Name the enzymes involved in somatic hypermutation?
Activation induced deaminase (AID) and DNA repair genes
Where are follicular dendritic cells present?
In Germinal centres
What do follicular dendritic cells capture Antigens via?
Fc receptors and Complement receptors
What oversees B cell affinity maturation?
Follicular dendritic cells
Describe how follicular dendritic cells oversee B cell affinity maturation
-Centrocytes that have undergone somatic hypermutation express mutated BCR on surface
-Centrocytes thus compete with each other for antigen on FDC and for signals from Tfh cell
-If mutated BCR binds Ag on FDC better than unmutated, it will present more efficiently and receive CD40 signal from Tfh
Describe the function of Follicular T helper cells
-Recently defined subset of CD4 Th cells
-Specialised to help B cells
-By secreting either TH1 or TH2 type cytokines
What is the role of CD40 signal?
CD40 signal via CD40L expressed on Tfh
-Protects centrocytes from apoptosis
-Inducing isotype switching (different cytokines induce different isotopes)
What is the function of Activation induced deaminase (AID)?
Induces DNA breakage forming new constant region of antibody joined without affecting existing VDJ region
Describe the different isotopes of immunoglobulins that different antigens produce
-Polysaccharides (TI) produce IgM
-Proteins (TD) produce IgG1 and IgG3 or IgG4
-Ag at mucosal surfaces induce IgA
-Some Ag elicit IgE
Why do we need immunological tolerance
-Without tolerance, the immune system would attack the body’s own healthy cells as self reactive species are developed
-Prevents overreactions to harmless substances
-Helps regulate immune responses, ensuring the body doesn’t damage itself too much
-Important for accepting beneficial transplants and microbes (eg microbiota)
Describe how random TCR gene rearrangement can lead to T cells expressing TCR that are tolerant
-A fail to recognise self MHC (useless), B recognise self MHC+self peptide (dangerous), C recognise MHC+other peptide (useful)
-A die by neglect (no positive selection survival signals), B and C are expanded by positive selection, then B are eliminated by negative selection
-Leading to only C surviving
Since not all self antigens (eg insulin) are expressed in the thymus, how are these antigens introduced to naive T cells?
-Autoimmune Regulator Protein is a transcription factor that allows the expression of many tissue-specific antigen in the thymus
-So negative selection of T cells that recognise these antigens occurs
Describe how random BCR gene rearrangement can lead to no autoreactive B cells
-Autoreactive B cells are negatively selected in bone marrow
-B cells get a second chance to rearrange self reactive BCR through receptor editing
What occurs when immature B cells bind to self antigens?
Anergy (downregulating til unresponsive) or death or editing
How may T lymphocytes be made anergic?
-Unstimulated macrophages do not deliver a costimulatory signal to T cells recognising a non bacterial antigen
Name some other mechanisms of immunological tolerance
-Immunological ignorance (Ag not presented at sufficient levels)
-Privileged sites (Ag hidden away from immune system or suppressive cytokines)
-No T cells = No B cell response
-Regulatory T cells and B cells
What transcription factor do T regulatory cells express?
Foxp3
Describe regulatory T cells
-CD4 T cell subset that suppresses immune responses
-Crucial for preventing autoimmune responses
-Arise in thymus from T cells with high affinity TCR fro self AG
What are the types of regulatory T cells?
Natural T reg (nTreg) - arising in thymus
Induced T reg (iTreg) - Induced in periphery
How do regulatory T cells work?
-Suppressing anti inflammatory cytokines (eg IL10 and TGFβ) that inhibit self reactive T cells
-Binding directly to cells through cell surface receptors, eg blocking costimulatory molecules on dendritic cells
What are the different types of effector CD4 T cells?
-TH1
-TH2
-TH17
-Treg + Breg
-TFH
What do regulatory B cells secrete?
IL10 cytokines
Describe the role of TH1 cells
-Activate macrophages via the secretion of cytokines (eg IFNγ)
-Express CD40L which binds to CD40 on macrophage (activating it)
-Can kill chronically infected macrophages through Fas ligand induced apoptosis
Describe the role of TH17 cells
-Secrete IL17 cytokines
-Recruits neutrophils early in fungal infections
What subtypes of CD cells can Treg cells be?
-CD4
-CD25
-CD8
What does IL10 inhibit?
APC function
Describe the role of TH2 cells
-Secrete IL4, IL5, and IL13
-Promoting responses mediated by eosinophils and mast cells
Describe what TH1 cytokines promote and inhibit in terms of T cell development
-Promote commitment to TH1
-Inhibit development of TH2 and TH17
Describe what TH2 cytokines promote and inhibit in terms of T cell development
-Promote commitment to TH2
-Inhibit development of TH1 and TH17
Describe what TH17 cytokines promote and inhibit in terms of T cell development
-Promote commitment to TH17
-Inhibit development of Treg
Describe what Treg cytokines promote and inhibit in terms of T cell development
-Inhibit TH1, TH2 and TH17 responses
How are Tregs significant in pregnancy?
Suppress activation of the immune system, allowing for successful placentation
What are polarised responses
The pathways of immune activation that the body chooses based on the type of threat it faces (eg prioritising TH1 cells)
Why are polarised immune responses important?
-Allows tailoring immune response to types of threats, maximising efficiency whilst minimising damage to the body
-Prevents autoimmunity (excessive TH1)
-Prevents allergies (excessive TH2)
-Allows pregnancy
Do effector T cells (ie not naive anymore) require costimulation?
No!
What has an effect on the type of effector mechanisms required?
-Cellular site of pathogen
-Type of pathogen
-Stage of infection
Name some sites where infection may occur
-Extracellular interstitial spaces (eg blood, lymph)
-Extracellular epithelial surfaces
-Cytoplasmic spaces
-Vesicular
Describe innate defence mechanisms
-Rapid
-Utilises barriers, the complement system, phagocytosis, NK cells, antimicrobial peptides
-Non specific
-Ineffective against many pathogens
Describe host defence mechanisms
-Acquired/adaptive defence mechanisms
-Utilises antibodies, cell mediated immunity
-Takes longer to develop but exhibits memory
-Enhances and focuses innate defences
What are TH1 CD4 cells active against?
-Active against intracellular pathogens
-Activates macrophages and stimulate cytotoxic T cells
What are TH2 CD4 cells active against?
-Active against extracellular pathogens
-Support antibody production, particularly class switching to IgE
-Activates eosinophils, basophils and mast cells
What are TH17 CD4 cells active against?
-Active against extracellular bacteria and pathogens
-Important in attractive inflammatory cells such as neutrophils
Name some gram positive bacteria
-Staphylococcus aureus
-Streptococcus spp
Name some gram negative bacteria
-Campylobacter
-Salmonella
-Shigella
-Haemophilus
-Neisseria
How do bacterial cell walls affect the immune response?
-Components eg LPS, peptidoglycan bind to Toll like responses on macrophages
Describe Toll like receptors
Binding of pathogen associated molecular patterns (PAMPs) to TLR can
-Promote inflammation
-Promote dendritic cell maturation
-Influence differentiation of T cells
-Activate B cells
Give some examples of molecules that bind to intracellular toll like receptors
-dsRNA
-ssRNA
-CpG DNA
Give some examples of molecules that bind to extracellular toll like receptors
-Diacyl lipopeptides
-Triacyl lipopeptides
-Flagellin
-LPS
Describe the role of antibodies in bacterial infection
-Opsonisation (bind Fc receptors in phagocytes)
-Complement activation (promote inflammation, opsonise, lyse using MAC)
-Bind to and neutralise toxins
-Bind to surface structures to prevent mucosal adherence
What do defects in terminal complement components lead to?
Susceptibility to Neisseria spp
What effector cell is used against bacteria surviving within phagocytes?
TH1
What is the difference between normal and activated macrophages?
-Activated macrophages are better at phagocytosis and killing
-More efficient antigen presenting cells
-May stimulate inflammation
What activates a macrophage?
-The binding of CD40 on a macrophages surface to CD40 ligand secreted by TH1 cell
OR
-IFNγ secreted by TH1 binding to its receptor on the macrophage cell surface
Give an example of the different outcomes of a disease (in this case leprosy) depending on types of immune response
-Tuberculoid leprosy develops from a strong TH1 response, containing the bacteria within the skin and nerves, limiting bacterial spread.
-Lepromatous leprosy develops from a weak TH1 response, allowing the bacteria to multiply and spread throughput the body, often due to STRONGER TH2 response
What are the innate defences against virus infected host cells?
Interferons (IFN⍺ and IFNβ) and Natural killer cells
What is the action of IFN⍺ and IFNβ secreted by virus infected host cells on healthy cells?
-Induces resistance to viral replication in all cells by inducing Mx proteins, 2’-5’ linked adenosine oligomers and the PKR kinase
-Increases MHC class I expression and antigen presentation in all cells
-Activate dendritic cells and macrophages
-Activate NK cells to kill virus infect cells
-Induces chemokines to recruit lymphocytes
What do natural killer cells and T cells do in response to a viral infection?
Secrete IFNγ which
-inhibits TH2 response and promotes TH1
-Recruits macrophages
How do natural killer cells kill virus infected cells?
Kill by extracellular mechanism using perforion and granzyme
How do Natural Killer cells distinguish between infected and uninfected host cells?
-Activated by carbohydrate ligand receptors, triggering lysis
-Inhibited by MHC class I molecules (only TCRs can bind)
What are the two mechanisms of cell mediated specific immunity to viruses?
-Cytotoxic T cells (CD8+) recognising viral peptide
-Cytokines with antiviral activity eg IFNγ
Describe the mechanisms by which Cytotoxic T cells may apoptosise a viral infected cell
-Secretion of cytotoxic granules (eg perforin, granzymes)
-Fas ligand binds to Fas receptors (CD95) on the cell surface of infected cells, triggering caspases
In influenza, what do antibodies recognise?
Viral haemagglutinin and Neruaminidase
What immune cell does HIV target?
CD4
How does our body control the HIV infection?
-Antibodies to HIV do not seem to protect against the virus
-Instead, patient with higher levels of Cytotoxic T cell activity show slower disease progression
How may antibodies protect against parasites?
-May opposonise
-Activate complement, leading to MAC formation and lysis
-ADCC (antibody dependent cell mediated cytotoxicity)
Name some methods that pathogens have developed to evade the immune response?
-Concealment of antigens
-Antigenic variation
-Immunosuppression
-Interference with effector mechanisms
What is the role of IgE antibodies in fighting parasites?
-Mast cell mediated inflammation
-Eosinophil antibody-dependent cell mediated cytotoxicity
Describe how pathogens may conceal antigens
-Viruses may inhibit antigen presentation by MHC Class I (eg HSV)
-May occupy immune privileged sites (eg chickenpox may develop into shingles)
-May uptake host molecules, as a disguise (eg schistosomes)
Describe how pathogens may use antigenic variation to evade the immune system
-They may have a large number of antigenic types (eg streptococcus pneumoniae)
-Pathogens may mutate frequently, undergoing small changes/antigenic drift and larger recombination events/antigenic shift
-Pathogens may switch on different genes at different times, producing different antigens, via genetic rearrangement
What antigen changes in trypanosome’s gene switching to avoid the immune system?
Variant specific glycoprotein
Describe how many antigens that Streptococcus pneumoniae vaccines must be active against?
23 capsule antigens
Describe methods that viruses suppress the immune system
-Pathogens may infect immune cells directly (eg HIV infecting CD4)
-Pathogens may induce regulatory T cells, secreting immunosuppressive cytokines (IL10)
Describe how pathogens may evade immune defences by interfering with effector mechanisms
-May secrete molecules that interfering with antibody function (eg IgA proteases secreted by Streptococcus pneumoniae)
-May secrete molecules interfering with complement (eg enzymes that break down C3a by pseudomonas)
-May secrete molecules by binding cytokines
-May secrete molecules that inhibit cytokine activity (eg IL10)
Describe how host responses may contribute to immunopathology
-Infections may cause chronic immune cell activation, leading to granulomas
-Infections may lead to cytokines storms, where excessive cytokines are released leading to a hyper inflammatory state, damaging tissues and organs
Describe the effects of Interferon gamma (IFNγ)
-Enhances expression of MHC class I, meaning more sites for TCR to bind and activate T cells
-NK cells are more likely to kill cells that lack MHC I
Name a method of natural active immunity?
Natural infection
Name a method of artificial active immunity?
Immunisation
Name a method of natural passive immunity?
Placental transfer of maternal IgG
Name a method of artificial passive immunity?
Human IgG transfusion
Describe passive immunity
-Short lived
-Hypogammaglobulinaemia in infants as maternal IgG declines
-examples of artificial is Tetanus antitoxin
Describe active immunity
-Exploits immunological memory
-Faster to develop, greater in magnitude, may be qualitatively better
Describe herd immunity
-Disease spread is limited as a significant portion of a population becomes immune to an infectious disease
-Based on that vulnerable people are protected as those who they interact with are immune
What are the main types of vaccines?
-Inactivated dead organisms
-Attenuated (live but virulence disabled)
-Subunit vaccines (protein fragments)
-Toxoid
-Conjugate something with low antigenic property with high
Give some requirements of an effective vaccine
-Safe
-High level of protection
-Long lasting protection
-Correct response
-Low cost
-Easy to administer
-Stable
Describe inactivated vaccines
-Made from viruses or bacteria that have been inactivated so they cannot cause disease
-Usually with heat, chemicals or radiation, so their structure is preserved
-Do not revert to virulent form, making them safe
-Require booster doses in most cases
Describe attenuated vaccines
-Made from live viruses or bacteria that have been modified to replicate slowly or not at all
-Does not cause illness but gives long lasting immunity
-However must be stored correctly, and not recommended for immunocompromised individuals
Describe subunit vaccines
-Contain only specific parts of a pathogen, typically proteins, sugars or other antigens
-Cannot cause disease and have a low risk of side effects
-Often requires booster doses
Describe toxoid vaccines
-Specific to bacteria that release harmful toxins, preparing for neutralisation
-Inactivated toxins using heat or chemicals
-Often require emulsion in aluminium solutions
Describe conjugate vaccines
-Protein carrier is linked to polysaccharide antigens so that this is more easily recognised in immunocompromised people.
-Converts TI-2 to a thymus dependent form
What is reverse vaccinology?
-Using genomic information to identify potential antigens directly from the DNA sequence of a pathogen
-Removes the pathogen cultivation in a lab aspect of vaccinology
-Useful for complex pathogens that have numerous strains
What are adjuvants?
-Substances added to vaccines to enhance the body’s immune response to the vaccine’s antigen
-Often aluminium in humans
In what area of blood are antibodies found?
In the plasma
What is blood serum?
Plasma once the blood clot has been removed
What is serum from an immunised person/animal known as?
Antiserum
What does blood antiserum contain?
-Antibodies that bind a certain Ag
-Other soluble blood components eg growth factors, proteins
-NO CELLS OR CLOTTING PROTEINS
Describe the antibodies found in antiserum
-Many different antibodies that bind the same antigen
-Different antibodies may bind different regions (epitopes) of the same antigen
How may different antibodies be purified from antiserum?
Using gel filtration chromatography or affinity chromatography
Simplify gel filtration chromatography
-Separates molecules based on size
-Uses column filled with porous beads, through which molecules of different sizes pass at different rates
Simplify affinity chromatography
-Separates molecules based on specific interactions between a target molecule and a ligand attached to a stationary phase (resin)
-Used to purify a certain substance
Give some weaknesses of using antiserum in immunisation
-Antibodies created will never be the same
-Short lived immunity, lasting only a few weeks or months
-Limited efficacy against mutating pathogens
-Does not induce memory
Describe the production of hybridomas
-Animal is IMMUNISED against an antigen
-B cells are HARVESTED and FUSED with MYELOMA cells (cancerous cells), which produce antibodies indefinitely
-Fused cells are placed in a SELECTIVE medium (HAT medium) which only allows hybridomas
-MONOCLONAL ANTIBODIES are produced
Describe labelled antibodies
-Labelled using an enzyme (HRP,AP) or a fluorochrome (PE, FITC)
-Allows confirmation of the presence of antigens
-Label does not affect antibody/Ag binding
What can you use antibodies for in research?
-Purifying biological molecules from a mixture using AFFINITY CHROMATOGRAPHY
-Identifying the location of a protein within a cell using IMMUNOFLUORESCENCE MICROSCOPY
-WESTERN BLOTTING
-ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
-FLOW CYTOMETRY
Describe secondary antibodies
-Secondary antibodies bind specifically to primary antibodies (which bind to the targeted antigen)
-This increases the sensitivity and reducing costs
Describe the types of ELISA
-Direct ELISA (detect antigens directly with an enzyme linked antibody)
-Indirect ELISA (Uses a primary antibody to bind the antigen and a secondary enzyme linked for detection)
-Sandwich ELISA (uses two antibodies for detection: a capture immobilising antigen and a secondary enzyme linked antibody)
Describe the steps of ELISA
-Plate wells are COATED with either an antibody or antigen
-Non specific sites are blocked to prevent background noise
-Sample containing the target antigen or antibody is added and allowed to bind
-Substrate specific to an enzyme is added, producing a detectable signal
-Colour intensity is measured using a spectrophotometer
Describe the basic principles of flow cytometry
-Forward light scattering indicate cell size, side light scattering indicate reflects cell granularity
-Cells are passed through a laser beam, and the intensity of forward and size scattering
Describe FACS (Fluorescence activated cell sorting) cytometry
Sorts cells based on fluorescent labelling, which target specific markers on the cell surface or within the cell
Describe how we can use FACS to determine which immune cell has been examined
-Antibodies specific for certain molecules (eg CD3, CD4, CD8) can be conjugated with fluorescent dyes
-and used to identify the number of antibody “positive cells” in a mixed population
Give the steps in FACS data analysis
-GATING select specifics cells based on FSC and SSC, reducing noise from dead or unwanted cells
-FLUORESCENCE COMPENSATION removes overlapping signal of multiple fluorophlores
-Use GATES to exclude doublets or clumped cells, ensuring only single cells are analysed
-IDENTIFY CELL POPULATIONS
-VISUALISE DATA
-QUANTIFY
Give the biological components that make up innate immunity
-Physical barriers (skin, mucous membranes)
-Immune cells (macrophages, neutrophils, dendritic cells)
-Complement system
-Inflammatory responses
Give the biological components that make up adaptive immunity
-Lymphocytes (T+B)
-Antibodies (produced by B cells)
-Antigen presenting cells (eg Dendritic cells)
Give the key differences in primary and secondary immune responses
-Primary Response: Slower, lower intensity, and involves the generation of memory cells. Initially IgM are produced, followed by IgG and others
-Secondary Response: Faster, stronger, with a more efficient and robust immune reaction due to the presence of memory cells. IgG are produced much faster and at higher levels
Give a summary of the three signals required to activate B cells (Signal, Source, Role)
Summary of the Three Signals
Signal Source Role
Signal 1: BCR Engagement, Antigen, Initial activation and antigen presentation.
Signal 2: CD40-CD40L Interaction, Th cell (CD4+), Costimulation for full activation.
Signal 3: Cytokines, Th cell (CD4+), Drives class switching and effector function.
What are plasmacytoid dendritic cells specialised for ?
Specialized for recognizing viral infections via Toll-like receptors (TLR7 and TLR9)
