Basic Principles - Teeling Flashcards
What is the hematopoietic stem cell?
All immune cells are derived from a common progenitor known as the hematopoietic stem cell (HSC)
The HSC develops into two progenitor types:
- Lymphoid
- Myeloid
Most lymphoid cells contribute to adaptive immunity, while most myeloid cells contribute to innate immunity.
What cells are under the lymphoid lineage?
B Cells
T Cells
NK Cells
These are the cells of the adaptive immune system
Responsible for fighting antibody/cell mediated immune responses
What cells are under the myeloid lineage?
Neutrophil
Eosinophil
Mast Cell
Basophil
Monocyte
- Dendritic cell
- Macrophage
These are the cells of the innate immune system
Responsible for first interaction with pathogens
Role and structure of the innate immune system?
Immediate response
Non Specific
No immunological memory
Humoral:
- Pattern receptors
- Complement
- Enzymes
- Cytokines
Cellular:
- Phagocytes
- Natural Killer Cells
Role of the adaptive immune system?
Long term
Specific to antigen
Lag time from exposure to response
Immunological memory after exposure
Humoral:
- Antibodies
- Cytokines
Cellular:
- T Cells
- B Cells
What is the role of the immune system?
Defence (of host) against threat of disease by pathogenic infectious organisms (pathogen)
IS complexity due to range of organisms encountered – evolved to deal with many challenges
IS protects against tumours (some)
Vaccines offer exciting new hopes for infections and cancer therapy
Chronic immune responses can cause disease, for example sepsis, autoimmunity, Type-2 Diabetes
What are some innate physical barriers?
Anatomic:
- Skin
- Mucous membranes
Physiological:
- Temperature
- Low pH
- Chemical mediators
What is inflammation?
It is a complex biological response to harmful stimuli like pathogens or injury
Swelling, redness, heat, pain and immobility
Fluids leak from blood vessels and collect in tissues
Leukocytes will also begin to exit from blood vessels to investigate the local tissue in a process known as extravasation
What are the signs of inflammation?
Cardinal signs of inflammation:
- Rubor (redness)
- Tumor (swelling)
- Calor (heat)
- Dolor (pain)
- Functio Laesa (loss of function)
What are PPR (Pattern Recognition Receptors)
PRRs are a class of proteins expressed by cells of the innate immune system to recognise specific molecular patterns associated with pathogens
They detect the presence of foreign pathogens or danger signals and initiate innate immune responses
Examples:
- Toll-like receptors (TLRs)
- NOD-like receptors (NLRs)
- RIG-I-like receptors (RLRs)
- C-type lectin receptors (CLRs)
What are PAMPs (Pathogen-Associated Molecular Patterns)
PAMPs are conserved molecular structures commonly found on pathogens but not on host cells
They are recognised by PRRs as signals of microbial invasion
Activation of PRRs by PAMPs triggers immune responses such as inflammation, phagocytosis, and the release of antimicrobial molecules
It also activates innate responses such as:
- Chemokines / cytokines to recruit cells
- Phagocytosis of pathogens
- Lysis of pathogens by antimicrobial peptides
What are DAMPs (Damage-Associated Molecular Patterns)
DAMPs are endogenous molecules released by damaged or dying host cells in response to tissue injury or stress
They alert the immune system to tissue damage and activate immune responses
DAMPs signal danger and trigger inflammation, repair processes, and the recruitment of immune cells to the site of tissue damage
What are TLR (Toll-like Receptors)
TLRs are a class of PRRs present on the surface or within cells of the innate immune system
They recognize specific PAMPs and DAMPs, initiating immune responses
They trigger an intracellular signalling cascade
Activation of TLRs leads to the production of cytokines, chemokines, andother molecules that promote inflammation and coordinate immune responses against pathogens.
What is the TLR signalling cascade in mammals?
MyD88 – Myeloid differentiation primary response gene (88) is a universal TIR adaptor (except TLR3)
MyD88 interacts via a TIR domain
MyD88 promotes association of IRAK1 & 4 kinases (interleukin-1 receptor-associated kinase)
IRAK4 phosphorylates IRAK1 creating a docking site for TRAF6 (TNF-receptor-associated factor 6)
TRAF-6-IRAK1 dimer complex dissociates
Complexes with TAK1 (+other proteins) (TGF-beta activated kinase 1) causing kinase activation
TAK1 is pivotal as it activates both NFkappaB and Map kinase pathways
Increased transcription of target genes
TAK1 activates IKK which phosphorylates IkappaB causing activation of NFkappaB
What does TLR activation in macrophages result in?
TLR activation in macrophages results in oxidative burst (phagocytosis) and inflammatory cytokine/chemokine release
What does TLR activation in dendritic cells result in?
TLR activation in dendritic cells results in maturation, antigen presentation, and cytokine production- bridge to adaptive immunity
What is a cytokine?
Small proteins released by cells
Have a specific effect on the interactions and communications between cells
Regulate the immune response
Can promote or inhibit inflammation
Are crucial in fighting infections and in immune responses to disease
What are natural killer cells?
Natural Killer cells are a type of lymphocyte (a white blood cell) in the immune system
They play a major role in the host-rejection of both tumours and virally infected cells
NK cells are known for their ability to kill without prior immunisation to the target
They release cytotoxic (cell-killing) granules that lead to the destruction of the target cell
Unlike T-cells, NK cells do not need antigen recognition to attack their targets
They are part of the innate immune system and provide a rapid response to virally infected cells and tumour formation
What are dendritic cells?
Dendritic cells are immune cells that form part of the mammalian immune system
They are antigen-presenting cells (APCs) which means they process antigen material and present it on the cell surface to the T cells of the immune system
Dendritic cells act as messengers between the innate and the adaptive immune systems
They have a unique ability to capture and present antigens, leading to the activation of T cells
Dendritic cells can be found in peripheral tissues such as the skin (where they are often referred to as Langerhans cells) and the inner lining of the nose, lungs, stomach, and intestines
They play a crucial role in initiating and modulating the immune response, especially in the context of infection, cancer, and vaccination
What are macrophages?
Macrophages are large white blood cells found in almost all tissues
They are part of the immune system and play a key role in the initiation, maintenance, and resolution of inflammation
Macrophages are involved in the detection, phagocytosis (engulfing and then digesting), and destruction of bacteria and other harmful organisms
They can also present antigens to T cells and initiate inflammation by releasing molecules known as cytokines
Macrophages play a role in wound healing and tissue repair by removing dead cells and stimulating the repair of damaged tissues
What do cytokines and interferons do?
Released from cell after immune response sensing and signal transduction pathway
They are able to:
- Signal neighbouring cells to ‘put up barriers’
- Signal infected cells to die
- Recruit white blood cells to stimulate long lasting immunity
What is the JAK-STAT pathway?
The JAK-STAT pathway is a signaling mechanism used by a group of cell surface receptors for cytokines and growth factors
It involves the activation of Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs)
This pathway is essential for transmitting information from extracellular signals to the cell nucleus, influencing gene expression
What are Janus Kinases (JAKs)?
JAKs have two functional sites:
- A binding site to associate with the cytokine R
- A catalytic site, which when activated, has tyrosine kinase activity
JAKs bind a receptor and tyrosine phosphorylation creates a binding site for the SHa domain of STATs, firstly on the receptor and then on the Stat itself- this leads to dissociation & dimerisation
WATCH THE VIDEO (QUICK AND SIMPLE)
What are Signal transducer and activator of transcription (STATs)?
It is a transcription factor (TF) that binds
DNA sequence-specifically and promotes transcription from GAS element in response to cytokine stimulation
STATs may operate with other TFs
WATCH THE VIDEO (QUICK AND SIMPLE)
Effect of IL-1Beta cytokine
Local Effects:
- Activates vascular endothelium
- Activates lymphocytes
- Local tissue destruction
- Increases access of effector calls
Systemic Effects:
- Fever
- Production of IL-6
Effect of TNF-alpha cytokine?
Local Effects:
- Activates vascular endothelium and increases vascular permeability, which leads to increased entry of IgG, complement, and cells to tissues and increased fluid drainage to lymph nodes
Systemic Effects:
- Fever
- Mobilisation of metabolites
- Shock
Effects of IL-6 cytokines?
Local Effects:
- Lymphocyte activation
- Increased antibody production
Systemic Effects:
- Fever
- Induces acute-phase protein production
Effects of CXCL8 chemokines?
Local Effects:
- Chemotactic factor recruits neutrophils, bascphils, and T calls to site of infection
No systemic effects
Effects of IL-12 cytokines?
Local Effects:
- Activates NK cals
- Induces the differentiation of CD4 T-cells into TH1 cells
What is the complement system?
It is a part of the immune system that enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane
It consists of a series (around 40) of small proteins found in the blood, generally synthesised by the liver, and within tissues throughout the body
These proteins work together in a cascade fashion, where the activation of one component leads to the activation of the next, resulting in a rapid response to threats
The system is part of the innate immune response but also bridges with the adaptive immune system
It can be activated through three main pathways:
- The classical pathway
- The lectin pathway
- The alternative pathway
Each initiated in different ways but converging on the same terminal pathway that leads to the destruction of pathogens
What is complement activation?
It is the process by which complement proteins are activated in the blood, leading to a series of immune responses aimed at defending against pathogens
Activation can occur via three pathways:
- The classical pathway (triggered by antibody-antigen complexes)
- The lectin pathway (initiated by mannose-binding lectin binding to pathogen surfaces)
- The alternative pathway (activates spontaneously on pathogen surfaces)
Upon activation, a series of proteolytic reactions ensue, resulting in the production of key components that enhance inflammation, opsonisation (marking of pathogens for destruction), and the formation of the membrane attack complex (MAC) that can lyse pathogen cell membranes
The activation cascade is tightly regulated by complement control proteins to avoid damage to host cells
The outcome of complement activation includes direct killing of pathogens, facilitation of phagocytosis, and enhancement of the inflammatory response
What is the compliments classical activation pathway?
- Stimulated by an antigen bound to an antibody (antigen/antibody immune complex)
- Interacts with C1q-C1r-C1s
- Then recruits further components
What is the compliment systems alternative activation pathway?
- Activated by direct presence of bacteria, fungi, virus
- C3 (protein in blood) is hydrolysed in presence of stimuli
- C3 changes conformation
- C3 recruits proteins B, D and P to form complex which is then cleaved into C3a
Merging of different compliment pathways
All pathways merge at the key event, the proteolytic activation of the central C3 to C3b
What are the basic functions of the compliment system?
- Lysis of cells, bacteria and viruses
- Opsonisation which promotes phagocytosis of antigen
- Binding to specific compliment receptors triggering cell activation
-Immune complex clearance
Neutrophil overview
Also called polymorphonuclear leucocyte (PMN)
Short lived cell
First line of defence – recruited to infection (IL-8, C5a)-chemokine
Detect and phagocytose pathogens
Effector mechanisms to kill pathogens (lysosomal killing mechanisms)
What are C3a and C5a known as, and what are their main functions in the complement system?
Known as anaphylatoxins
Potent inflammatory mediators
Increase vascular permeability, leading to edema
Attract and activate phagocytes (neutrophils, macrophages)
C5a is particularly potent in attracting neutrophils
What role does C3b play in the complement system?
Functions as an opsonin
Binds to pathogen surfaces, marking them for destruction (opsonization)
Involved in the amplification loop of the alternative pathway
How does C4b contribute to the complement system?
Plays a role in the classical and lectin pathways
Binds to surfaces and forms a complex with C2a to create the C3 convertase
C3 convertase cleaves C3 into C3a and C3b
What is the role of C5b in the complement system?
Initiates the assembly of the membrane attack complex (MAC)
Associates with C6, C7, C8, and multiple C9 molecules
Forms the MAC, which creates pores in the pathogen cell membrane, leading to lysis and death
What is neutrophil extravasation and describe its steps?
Neutrophil extravasation is the recruitment of neutrophils from the blood stream into tissues
Chemokines at vessel wall cause chemoattraction of the receptors on the neutrophil towards the infection site
The neutrophil will then interact with the vessel wall and start rolling over the cells
They do this as they are able to interact with receptors that are either expressed on the neutrophil or expressed on the endothelial cells
Endothelial cells express selectins (adhesion molecules)
Neutrophils express a glycan (in leukocytes, it is a sialyl lewis x glycan)
The glycans are able to recognise the selectin and able to bind and cause the slowing down of the neutrophil
As they are slow, they can then move through gap junctions in the cells
How does tethering and rolling occur in neutrophil migration?
Neutrophils express glycan structures (sialyl lewis x as pictured) which are able to interact with either P or E selectins
Neutrophils also express integrins called LFA-1, which also interacts with the selectins
This leads to transient adhesion and slowing down of the neutrophils
How are the neutrophils ‘stopped’ in neutrophil migration
Chemokines that are expressed at the site of infection are able to activate the neutrophil
The activation of the endothelial cells increases the expression of adhesion molecules such as ICAM-1
Conformation change of the LFA-1 integrins on the activated neutrophils allows tight binding to ICAM-1 (adhesion molecule) on the endothelial cells
This causes firm adhesion and arrest of the neutrophil
How do neutrophils undergo extravasation in neutrophil migration?
After stopping, the neutrophil squeezes between endothelial cells into tissue attracted by chemokines at the site of infection
What are some types of tissue macrophages?
Kupffer cell (liver)
Microglia (brain)
Osteoclast (bone)
Alveolar macrophage (lung)
All play an important role in maintaining homeostasisB
Basic macrophage mechanism?
Chemotaxis and adherence of microbe to phagocyte
Ingestion of microbe by phagocyte
Formation of a phagosome
Fusion of the phagosome with a lysosome to form a phagolysosome
Digestion of ingested microbe by enzymes
Formation of residual body containing indigestible material
Discharge of waste materials
What are the mechanisms of a macrophage that destroy the pathogen?
Acidification:
- pH of 3.5-4.0 reduces bacterial function
Toxic oxygen-derived products:
- Creates highly reactive oxygen species that are damaging to for pathogens (also damages host)
Toxic nitrogen oxides:
- Nitric oxide NO
Antimicrobial peptides:
- Defensins and cationic proteins
Enzymes:
- Lysozyme - Dissolves cell walls of some gram positive bacteria
- Acid hydrolases - Further digest bacteria
Competitors:
- Lactoferrin (binds Fe) and Vitamin B12- binding protein
B cells overview
Humoral Immunity
B cells arise in the bone marrow of adult mammals
B cells recognize native, (extracellular) antigen via BCR
B cells produce antibodies and cytokines
T Cells overview
Cell-Mediated Immunity
T cells arise in the bone marrow but mature in the thymus.
They do not produce antibody molecules but have surface receptors that are structurally related to Ig (TCR)
T cells recognise peptide fragments of antigens complexed with MHC glycoproteins (Antigen presenting cells)
CD4+ T Cell overview?
CD4+ T cells, also known as helper T cells, play a critical role in orchestrating the immune response by helping other cells of the immune system respond to pathogens
They are involved in activating and directing other immune cells, such as B cells, other T cells, and macrophages
Receptors:
- Αβ TCR, CD3, CD4
Secretions:
- IFN-γ, IL-2, IL-4, IL-5, IL-13, IL-10 (cytokines)
Functions:
- Immunity against intracellular bacteria and parasites
- Provide help to B cells and CD8 T cells
- Promote humoral immune responses
CD8+ T Cell
CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), are primarily responsible for killing infected cells and cancer cells
They recognise and destroy cells infected with pathogens and cells that have become cancerous
Receptors:
- Αβ TCR, CD3, CD8
Secretions:
- IFN-γ, perforin, granzyme
Functions:
- When activated they become cytotoxic
- Kill virally infected cells
- Kill tumour cells
How do antibodies and T-cell receptors differ in the way they recognize antigens?
Antibodies bind directly to epitopes on the surface of antigens
T-cell receptors (TCRs) do not recognise free-floating antigen
Instead, they recognise epitopes (part of an antigen that is recognised by the immune system) that are often hidden and must be presented on MHC molecules
What steps are involved in the processing of antigens for T-cell recognition?
The antigen is internalised by an antigen-presenting cell (APC)
It is then broken down into peptide fragments
The epitope peptide binds to self molecules known as Major Histocompatibility Complex (MHC) molecules
What is the role of MHC molecules in T-cell recognition?
MHC molecules present the epitope peptides on the surface of the antigen-presenting cell
T-cell receptors recognise the complex formed by the MHC molecule and the epitope peptide
T cell receptor structure
Heterodimer composed of an α and a β chain
Each chain has two domains, one variable and one constant domain
How do TCR produce a signal?
The TCR associates with CD3 molecules
CD3 allows a signal to be fed into the cell
TCR specificity
Each T cell has a specific cell surface receptor (TCR)
Specificity is determined prior to exposure to antigen
There are >10^12 different T cell clones
There are only limited number of genes that encode TCRs
How do T cells generate diversity to recognise many pathogens?
Answer lies in VDJ (variable–diversity–joining) recombination
Many genes that make up the TCR locus
These genes rearrange to make many receptors
The recombination occurs at recombination signal sequences (RSS)
It is catalysed by enzymes called VDJ recombinase
The most important are RAG1 and RAG2
Describe the process of VDJ recombination in T cells
VDJ recombination is the process of rearranging variable (V), diversity (D), and joining (J) gene segments
This recombination generates diverse T cell receptors (TCRs) that are essential for the immune response
Occurs at the TCR alpha (α) and beta (β) loci during T cell development in the thymus
What is the function of recombination signal sequences (RSS) in VDJ recombination?
RSS are short DNA sequences that flank V, D, and J gene segments
Serve as recognition sites for the recombination machinery
Consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs, following the 12/23 rule
What enzymes are used in VDJ recombination?
Enzymes known as VDJ recombinase
Most important are RAG1 and RAG2
These are expressed in lymphocytes
How does VDJ recombination generate diversity?
Combinational diversity:
- From the different combinations of gene segments
Junctional diversity:
- From the addition of nucleotides when recombination occurs
What are the steps of VDJ recombination?
Recognition of Signal Sequences:
- VDJ recombination is initiated at specific DNA sequences called recombination signal sequences (RSS)
- The RSS is recognized by the recombination activating gene (RAG) complex
Cleavage by RAG Complex:
- The RAG complex, composed of RAG1 and RAG2 proteins, binds to the RSS
- It cleaves the DNA to form hairpin structures at the ends of the V, D, and J segments
Hairpin Opening:
- The hairpin structures formed are opened by the RAG complex with the help of Artemis, making the ends of DNA single-stranded (P-nucleotides)
Addition of N-Nucleotides:
- Terminal deoxynucleotidyl transferase (TdT) adds random nucleotides (N-nucleotides) at the junctions, increasing diversity.
Joining of Segments:
- The ends are then brought together and repaired by DNA repair enzymes to form a coding joint, completing the recombination process
What are major histocompatibility complexes MHC?
MHC = Major Histocompatibility Complex
MHC molecules bind to proteins from viruses and bacteria and present them to T cells
They also bind to self peptides from the cell
This is called Antigen Presentation
Class 1 vs Class 2 MHC
Class I MHC
- Found on most nucleated cells (not RBC)
- Present endogenous antigens (intracellular, internal)
- Display self proteins, virus proteins, intracellular pathogens
- Present antigen to cytotoxic T cells (CD8)
Class II MHC
- Found primarily on Antigen Presenting Cells
- Present exogenous antigens (extracellular, external)
- Phagocytosis, receptor mediate endocytosis
- Present antigen to helper T cells (CD4)
How are naive T cells activated against a pathogen?
Dendritic cells take up pathogens and traffic to the lymph node
In the lymph node they interact with naive T cells – activating T helper cells and killer T cells
How are naive CD4+ T cells activated
Three signals are involved in activation of naïve CD4+ T cells by APC’s
1) MHCII + foreign peptide
2) Co-stimulation signal (CD28-B7)
3) cytokines
How do CD28 and CTLA-4 regulate T cell activation and downregulation?
CD28:
- Initiates T cell activation by binding to B7 molecules on APCs
- Necessary for the second signal in T cell activation, leading to IL-2 production and T cell proliferation
CTLA-4:
- Counteracts CD28’s actions by binding to B7 with higher affinity
- Leads to T cell down regulation and tolerance, preventing over activation
How do various cytokines influence T helper cell differentiation? (T helper subsets)
Th1 Cells:
- Differentiation driven by IFN-γ and IL-12
- Produce IFN-γ, which activates macrophages and promotes cell-mediated immunity
Th2 Cells:
- IL-4 drives differentiation
- Produce IL-4, IL-5, IL-9, and IL-13, which are important for humoural immunity and defense against extracellular parasites
Th17 Cells:
- Differentiation requires IL-6 and TGF-β; IL-23 stabilises the subset
- Produce IL-17 and are involved in defense against extracellular bacteria and fungi, as well as in inflammatory and autoimmune diseases
Treg Cells (Regulatory T Cells):
- Differentiation is driven by TGF-β
- Produce IL-10 and TGF-β, which suppress immune responses and inflammation
How are naive CD8+ T cells activated
CD8 T cell activation requires additional help
CD4 effector T cells amplify the activation of naïve CD8 T cells by further activating the APC
B7 expressed by DCs first activates the effector CD4 T cells to express IL-2 and CD40L
CD40L binds CD40 on the DC, delivering an additional signal that increases the expression of B7 and 4-1BBL by DCs, which in turn provides additional co-stimulation to the naïve CD8 T cell
The IL-2 produced by the activated CD4 T cells also acts to promote effector CD8 T cell differentiation
What do CD8 T cells kils?
They kill virally infected cells
CD8 T cells interact with APCs and detect pathogen peptides on MHC molecules
These T cells then become activated
Differentiate into cytotoxic T cells
How do CD8+ t cells kill?
Perforin:
- Aids in delivering contents of granules into the cytoplasm
Granzymes:
- Serine proteases, initiate apoptosis once injected into cytoplasm
Granulysin:
- Antimicrobial
What are the types of antigen presenting cells?
Three types of ‘professional’ APCs that express both MHC-I and MHC-II:
- Dendritic cells
- Activated macrophages
- Activated B cells
Cells other than APCs that express MHC-I are ‘target’ cells:
- Target can be virus infected, malignant and aging cells, or cells from a graft
Dendritic cells overview
- Detect danger signals at site of infection
- Transport antigen to lymph node
- Present antigen to naïve T cell
- Uses both MHC-I and MHC-II
- Major role: activation of naïve T cells
Activated macrophages overview
- Detects danger signal at site of infection
- Major role: present antigen to T cells that
arrive at the site of infection - Re-stimulation of T cells: keep the response going
Activated B cells overview
- B cells must be activated by Th cells to act as APCs
- BCR has very high affinity for antigens
- Major role: late in initial infection or early in subsequent infection by the same pathogen
Where does T cell development take place?
T cell development occurs in the thymus
Antibody structure
General Structure
- Y-shaped molecule
- Two identical heavy (H) chains
- Two identical light (L) chains
Regions
- Variable (V) region: Antigen-binding site
- Located at the tips of the Y-arms
- Determines antigen specificity
- Constant (C) region: Effector function
- Forms the base of the Y
- Dictates the antibody’s class
Chains
- Heavy chains: Define isotype (IgG, IgM, etc.)
- Light chains: Two types (kappa and lambda)
Domains
- Immunoglobulin (Ig) domains: Structural units
- Found in both V and C regions
Disulfide Bridges
- Stabilize the antibody structure
- Connect H chains together and H to L chains
Flexibility
- Hinge region: Allows flexibility
- Facilitates binding to multiple antigens
What happens during a B cell immune response?
Activation:
- B cells are activated by encountering their specific antigen
- They recognize antigens through membrane-bound immunoglobulins (B cell receptors or BCRs)
- Activation often requires help from T helper cells, which interact through the antigen presented by
- Major Histocompatibility Complex II (MHC II) on B cells.
Differentiation:
- Once activated, B cells can
differentiate into either plasma cells or memory B cells
Plasma cells:
- Secrete large amounts of antibodies specific to the antigen.
- These antibodies help neutralize pathogens and mark them for destruction by other immune cells.
- Plasma cells are primarily involved in the immediate defense during an infection.
Memory B cells:
- Do not secrete antibodies immediately.
- Persist in the body for years or even decades.
- Rapidly respond to subsequent exposures to the same antigen by quickly differentiating into plasma cells or proliferating.
- Provide long-lasting immunity and are the basis for the effectiveness of vaccines.
Specificity of B cells?
Each B cell has a specific antibody as a cell surface receptor (BCR)
Specificity is determined prior to exposure to antigen
There are >10^9 different antibody molecules, thus 109 different B cell clones
Only limited number of genes that encode antibodies
How is antibody diversity achieved?
There are three different genetic loci that make up the light chains and heavy chains
The genes that make up antibody molecules come in different segments
These segments are not next to each other in the genome
Segments are joined in different combinations to generate antibody diversity
This occurs through VDJ recombination
Recombination occurs at Recombination Signal Sequences (RSSs)
RSSs are made up of a heptamer – 12/23 - nonamer
How does VDJ joining occur?
VDJ joining occurs by DNA recombination
Ig genes rearrange by looping out intervening DNA.
First heavy chain D-J , followed by V-DJ, then light chain V-J
RAG1/2: unable to initiate recombination
SCID: unable to join correctly
What are the number of gene segments in human immunoglobulin loci
B cell development and maturation
Very early on B cells express BCR
Heavy chain is the first part of the BCR that needs to be generated and tested for specificity
First functional heavy chain prevents rearrangement of the other chromosome in a process called allelic exclusion designed to prevent expression of two antibodies by the same B cell
The same is true for light chain
Cells exit bone marrow as naïve, IgM/IgD positive B cells with as single antigenic specificity
B cell generation of specificity and diversity summary
BONE MARROW
- Antigen independent:
- Paring of different heavy and light chain
- Recombination of V, D and J segments
- Variability on the joins of the recombined gene segments
- P- and N region nucleotide addition
PERIPHERY
- Antigen dependent:
- Somatic hypermutation
- Class switching and affinity maturation
What is somatic hypermutation?
Further diversifies antibodies to generate a more specific antigen response
Introduces point mutations in the V region of the light and heavy chain
AID is a cytidine deaminase that introduces nicks in the DNA that are ‘repaired’
Requires a single strand of DNA
Targets DNA that is being transcribed ei. Ig genes
Somatic hypermutation allows for generation of higher affinity BCRs
B cell activation overview?
After exit from bone marrow, B cell activation,
proliferation and differentiation occurs in the periphery
and requires antigen
In the absence of antigen induced activation, B cells have a
short life span
B cell can be activated by two different pathways
- Thymus dependant (The B cell requires T helper cells)
- Thymus independent (No T helper cells required)
T-cell help is required for B cells that respond to peptide
antigens, but not for polysaccharide antigen
B cell activation
Antigen cross-links membrane bound Ig, generating signal, which leads to increased expression of class II MHC and co-stimulatory B7
Antigen-antibody complexes are internalised by receptor-mediated endocytosis and degraded to peptides, some of which are bound by class II MHC and presented on the membrane as peptide-MHC complexes
TH cell recognises antigen-class I| MHC on B-cell membrane. This plus co-stimulatory signal activates Th cell
- Th cell begins to express CD40L
- Interaction of CD40 and CD40L provides second signal
- B7-CD28 interactions provide co-stimulation to the Th cell.
- B cell begins to express receptors for various cytokines
- Binding of cytokines released from Th cell in a directed fashion sends signals that support the progression of the B cell to DNA synthesis and to differentiation
Where does B cell proliferation occur?
B cell proliferation occurs in the germinal centers of the lymph nodes
Lymph node structure
Primary Lymphoid Follicle:
- Small, solid, spherical structures without a germinal centre; predominately contains inactive B cells.
Secondary Lymphoid Follicle:
- Contains a germinal centre; primarily active in immune response, where B cells proliferate and differentiate.
Germinal Center:
- Central area in secondary follicles where B cells rapidly divide and differentiate into plasma cells or memory B cells.
High Endothelial Venule (HEV):
- Specialised blood vessels that enable lymphocytes to enter the lymph node from the bloodstream
T-Cell Area (Paracortex):
- Area adjacent to the follicles, rich in T cells and important for initiating immune responses
Medullary Cords:
- Extensions of lymphatic tissue within the medulla that contain B cells, T cells, and plasma cells
Efferent Lymphatic Vessel:
- Carries lymph, now filtered of antigens, out of the lymph node to the venous circulation.
B Cell route through lymph node?
Naive B cells travel to the lymph node via the bloodstream and leave via the efferent lymph
B cells that encounter antigen at the T-cell-B-cell border become activated
They form primary foci in the medullary cords
Some cells then migrate to the primary follicle, forming a germinal centre
Plasma cells migrate to the medullary cords or leave via the efferent lymphatics
Plasma cells migrate to the bone marrow
How long does it take for germinal centres to arise?
It takes roughly 7-10 days for germinal centres to arise after exposure to an antigen
What is the major outcome of the germinal center?
It is to generate higher affinity B cells from B cells of lower affinity
In general, affinity maturation and memory cell formation require germinal centers, however, some class switching and significant plasma cell formation occur outside germinal centers
What 3 differentiation events take place in germinal centres?
Three important B cell differentiation events take place in germinal centres:
- Affinity maturation
- Class Switching
- Formation of plasma cells and memory B cells
What are the types of antibodies in humans?
There are five main classes of antibodies (immunoglobulins) in humans, which are distinguished based on the type of heavy chain they contain. These classes are:
IgM: Often the first antibody produced in response to an infection; primarily found in blood and lymph fluid
IgD: Functions mainly as a receptor on B cells that have not been exposed to antigens
IgG: The most common type of antibody found in the circulation; important for virus and bacteria defense, and is the only antibody that can cross the placenta to provide protection to the fetus
IgE: Involved in allergic responses and defense against parasitic infections
IgA: Primarily found in mucous membranes lining the respiratory and gastrointestinal tracts, as well as in saliva and tears
Within the IgG class, there are four subclasses named IgG1, IgG2, IgG3, and IgG4. Each subclass is encoded by a different heavy chain gene (Gamma1, Gamma2, Gamma3, and Gamma4, respectively) and differs in its biological properties and roles in the immune response.
Rewatch 20th feb lecture from 24 mins to 32 mins
What molecules drive the differentiation of each type of antibody (IgM, IgD, IgG, IgE, IgA)?
IgM Differentiation
- Molecules: BAFF, APRIL, Activation through BCR
- Key Info: Early class switching, initial immune responses
IgD Differentiation
- Molecules: Co-expression with IgM during B cell development
- Key Info: Less defined role, part of B cell maturation
IgG Differentiation
- Molecules: IL-21, IFN-γ, TGF-β, CD40L-CD40 interaction
- Key Info: Promoted by Th1 cells, important for bacterial and viral defence
IgE Differentiation
- Molecules: IL-4, IL-13
- Key Info: Driven by Th2 cells, crucial in allergic responses and parasitic infections
IgA Differentiation
- Molecules: TGF-β, APRIL, BAFF
- Key Info: Supported by mucosal environment, important for gut and respiratory tract defense
