ABS Immunology

MCD Immunology Alexandra Burke-Smith

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1. Introduction to Immunology Professor Charles Bangham ([email protected])

1. Explain the importance of immunology for human health.

The immune system What happens when it goes wrong?

persistent or fatal infections

allergy

autoimmune disease

transplant rejection What is it for?

To identify and eliminate harmful non-self microorganisms and harmful substances such as toxins, by distinguishing self from non-self proteins or by identifying danger signals (e.g. from inflammation)

The immune system has to strike a balance between clearing the pathogen and causing accidental damage to the host (immunopathology).

Basic Principles

The innate immune system works rapidly (within minutes) and has broad specificity

The adaptive immune system takes longer (days) and has exisite specificity

Generation Times and Evolution Bacteria- minutes Viruses- hours Host- years The pathogen replicates and hence evolves millions of times faster than the host, therefore the host relies

on a flexible and rapid immune response

Out most polymorphic (variable) genes, such as HLA and KIR, are those that control the immune system, and these have been selected for by infectious diseases

2. Outline the basic principles of immune responses and the timescales in which they occur.

IFN: Interferon (innate immunity)

NK: Natural Killer cells (innate immunity)

CTL: Cytotoxic T lymphocytes (acquired immunity)


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Innate Immunity Acquired immunity

Depends of pre-formed cells and molecules Depends on clonal selection, i.e. growth of T/B cells, release of antibodies selected for antigen specifity

Fast (starts in mins/hrs) Slow (starts in days)

Limited specifity- pathogen associated, i.e. recognition of danger signals

Highly specific to foreign proteins, i.e. antigens

Cells involved: - Neutrophils (PMN) - Macrophages - Natural killer (NK) cells

Cells involved : - T lymphocytes - B lymphocytes - Dendritic cells - Eosinophils - Basophils/mast cells

Soluble factors involved - Acute-phase proteins - Cytokines - Complement

Soluble factors involved - Antibodies

Stimulates the acquired immune response

Innate Immunity

Anatomical barriers

- Skin as a mechanical barrier- keeps out 95% of household germs while IN TACT

- Mucus membrane in respiratory and GI tract traps microbes

- Cilial propulsion on epithelia cleans lungs of invading microorganisms

Physiological barriers

- Low PH

- Secretion of lysozyme, e.g. in tears

- Interferons

- Antimicrobial peptides

- Complement; responsible for lysing microorganisms

Acute-phase inflammatory response An innate response to tissue damage

Rise in body temperature, i.e. the fever response

This is followed by increased production of a number of proteins (acute-phase proteins), mainly by the liver.

Includes:

- C-reactive protein

- Serum amyloid protein

- Mannan-binding lectin

C-reactive protein and serum amyloid protein bind to molecules found on the cell wall of some bacteria and

fungi- pattern recognition

Mannan-binding lectin binds to mannose sugar molecules which are not often found on mammalian cells

These molecules are non-specific, but direct phagocytes e.g. macrophages to identify and ingest the

infectious agent

Cytokines

Small proteins that carry messages from one cell to another

E.g. to stimulate activation or proliferation of lymphocytes

kick-startacquired immune response

Send messages to other cells, e.g. to kill or secrete


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Cells of the innate immune system

Granular leukocytes Natural Killer (NK) cells

- Identify and kill virus-infected and tumour cells

- Complex recognition system- recognise HLA molecule of virus infected cell or tumour, and kill them

Macrophages

- Mononuclear phagocytes

- To main functions:

1. garbage disposal

- 2. Present foreign cells to immune system

Granulocytes

Neutrophils Eosinophils Basophils

Poluymorphonuclear neutrophils (PMN): multi-lobed nucleus

Bi-lobed nucleus

50-70% of circulating WBC 1-3% of circulating WBH


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- Bivalent- two identical antigen binding sites IgM - 10% of total serum Ig - Complex of 5 linked bivalent monomeric antibodies, therefore 10 identical binding sites- multivalent - Star-like shape - Important in primary immune response - Slightly lower affinity to antigens compared to IgG, which is compensated for by number of binding sites

IgA - 2 basic monomers; dimer with secretory piece - Found in body secretions, e.g. mucus membranes in GI tract - Contains a secretory component which protects it from digestive enzymes

IgE - Involved in allergic response and the response to helminths - Binds to basophils and mast cells - Triggers release of histamines

IgD - Complete function not known

A particularly antibody recognizes an antigen because that antibodys binding site it complementary to the EPIPTOPE (region approx 6 amino acids long) on the antigen. This forms the basis of the specificity of antigen recognition. How does an antibody kill a virus? Four important mechanisms:

1. Binds to the virus and prevents attachment to the cell

2. Opsonisation: virus-antibody complex is recognised and phagocytosed by macrophage

3. Complement- mediated lysis of enveloped viruses: cascade of enzymes in the blood which leads to the

destruction of cell membranes, and the destruction of the viral envelope

4. Antibody-dependant cell-mediated cytotoxicity (ADCC) mediated by NK-like cells (see earlier for explanation)

Cells of the acquired immune system

Lymphocytes

Agranular leukocytes

20-40% of the circulating WBC

99% of the cells in lymphatic circulation

T (thymus-derived) cells - Helper T cells: recognize antigen, help B cells to make antibodies and T cells to kill - Cytotoxic T cells: poisonous to cells,kill cells infected by viruses and intracellular bacteria

B (bone marrow-derived) cells - Make antibodies - Have insoluble antigen-binding receptor on its surface. In fact have multiple clones of this receptor;

monoclonal antibodies

NK (natural killer) cells - See earlier in notes

Each subset has distinct cell-surface molecules, e.g. CD4 on helper T-cell which is the receptor for HIV molecules

Lymphocyte precursors are produced in the haematopoietic tissue in the bone marrow


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T cells are then transported to the thymus, where they undergo THYMAL EDUCATION. Here 95-99% get destroyed as they have the potential to recognise host cells

4. Outline the role of clonal selection in immune responses.

Lymphocyte antigen receptors

B cell antigen receptor is a membrane-bound antibody, i.e. surface immunoglobulin which binds intact

antigens; recognises surface of protein, therefore antigen must be in native conformation

Expressed on the T cell surface are 2 protein chains (alpha and beta) which together make the t cell antigen

receptor (TCR). This binds to digested antigen fragments.

Each antigen receptor binds to an epitope on a different antigen, and is unique to a cell. There are many

copies of the receptor on the cell surface

The T-cell antigen receptor (TCR)

Recognizes complex of antigen peptide and HLA (MHC) molecule

HLA (Human leukocyte antigen) binds to little fragments of the pathogen, transports them to the surface so they can be recognized, e.g. so a virus cannot hide inside a host cell. Combination of short peptide from microorganism + HLA = recognition by TCR

MHC denotes the Major Histocompatibility Complex (also known as HLA) Generation of clonal diversity in lymphocytes

During B and T cell development, random genetic recombinations occur within each cell among multiple copies of immunoglobulin genes (B cells) or TCR genes (T cells). There are parallel genes, but they undergo random splicing and recombination which leads to a large repertoire of antigen receptors

These processes generate the diversity of clones of lymphocytes: each clone is specific to a different antigen. Primary Immune Response: clonal selection

A typical antigen is recognized by 1 in ~105 naive T cells

98% of T cells are in the lymph circulation and organs; 2% in blood.

Antigen binds to surface receptor on the B cell (Ig) or the T cell (TCR) and causes selective expansion of that clone.

The receptors which bind with highest affinity to the antigen are selected for, outcompete the other receptors , proliferate and survive to form effector lymphocytes

What happens when the antigen is removed?

Most lymphocytes that have proliferated recently will die after fulfilling their function (involves 2 or 3 mechanisms)

Some survive as memory cells. These are epigenetically modified so that next time the host is infected, the frequency of the receptors will increase.

How does the immune response clear a pathogen?


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Cytotoxic T lymphocytes (CTLs) kill cells infected by viruses or intracellular bacteria. It recognizes antigen peptide and HLA complex, releases granules of enzymes including proteases which digest DNA. The cell is therefore destroyed- APOPTOSIS

Antibodies bind to pathogens: the complex is destroyed or ingested by cells.

5. Understand the role of the physical organization of the immune system in its function.

How does a T cell meet its antigen?

Antigens are taken up by specialized ANTIGEN-PRESENTING CELLS (class of cells which are capable of taking

up particles, ingesting them and presenting proteins on their surface)

transported from the tissues into secondary lymphoid organs, where they meet T cells

initiate the acquired immune response

Antigen-presenting cells include B lymphocytes, macrophages and dendritic cells (which are most efficient)

Lymphoid Organs

Organized tissue in which lymphocytes interact with non lymphoid cells

Sites of initiation and maturation of adaptive immune responses.

Primary lymphoid organs produce the lymphocytes, e.g. bone marrow and thymus

Secondary lymphoid organs include lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT)

Lymphocytes and antigen-presenting cells circulate continuously blood and lymphatic vessels from

tissues via lymph nodes/spleen into the blood

T cells spend around 1-2 hours in the blood, but the rest of the day in the lymph

The tissues are patrolled by lymphocytes, antibodies and antigen-presenting cells.

For example, the skin contains lymphatic vessels that drain into local lymph nodes.

Gut lymphoid tissue controls responses in the intestinal tract.

Antigens present in the blood are taken to the spleen.


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Definitions Lymphocytes are mononuclear cells which are part of the leukocyte (white blood) cell lineage. They are subdivided into B (Bone marrow-derived) and T (Thymus-derived) lymphocytes. Lymphocytes express antigen receptors on their surface to enable recognition of a specific antigen Nave lymphocytes have never encountered the antigen to which their cell surface receptor is specific and thus have never responded to it. Memory lymphocytes are the products of an immune response, enabling the specificity of their specific receptor to remain in the pool of lymphocytes in the body. Innate immunity An early phase of the response of the body to possible pathogens, characterized by a variety of non-specific mechanisms (e.g. barriers, acids or enzymes in secretions) and also molecules and receptors on cells which are Pattern Recognition Molecules which recognize repeating patterns of molecular structure found on the surface of microorganisms. The innate immune response does not generate memory. Adaptive immunity is the response of antigen-specific lymphocytes to antigen, and includes the development of immunological memory. Adaptive responses can increase in magnitude on repeated exposure to the potential pathogen and the products of these responses are specific for the potential pathogen. Also known as Specific Immunity or Acquired Immunity. Active Immunity is the induction of an immune response by the introduction of antigen. Passive Immunity is immunity gained without antigen induction i.e. by transfer of antibody or immune serum into a nave recipient. Primary Response is the response made by nave lymphocytes when they first encounter their specific antigen. Secondary Response is the response made by memory lymphocytes when they re-encounter the specific antigen. T cells originate in the thymus. They recognize antigen presented at the cell surface by MHC/HLA molecules. Surface markers on T cells are CD3, CD4 & CD8 B cells originate in the bone marrow. They recognize free antigen in the body fluids. Surface markers associated with B cells are CD19, surface immunoglobulin class II MHC


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2. Immune Cells and Organs Dr Keith Gould ([email protected])

Primary lymphoid organs (thymus & bone marrow) for production of lymphocytes

Secondary lymphoid organs help antigen to come into contact with lymphocytes expressing appropriate specific receptors

Lymphocyte numbers are carefully regulated, and they recirculate

T cells express CD3, and recognise processed antigen presented by MHC molecules

B cells express CD19 and CD20, and recognise intact, free antigen

Important APC are dendritic cells, B cells, and macrophages

1. Name the primary and secondary lymphoid organs and briefly differentiate between their functions.

Primary lymphoid organs: organs where lymphopoeisis occurs, i.e. where lymphocytes are produced, including the

bone morrow and thymus to produce T and B lymphocytes.

Secondary lymphoid organs: where lymphocytes can interact with antigen and with other lymphocytes, including

spleen, lymph nodes, mucosal associated lymphoid tissues (MALT)

2. Draw simple diagrams to illustrate the structure of the thymus, lymph node, spleen, Peyers patch and

indicate the changes that occur after stimulation by antigen.

Primary lymphoid Organs:

Bone Marrow

- Site of haematopoesis, i.e.

generation of blood cells

- In an embryo, this happens in

amniotic sac

- In foetus, occurs in all bones, liver

and spleen. Marrow is also very

cellular

- In adults, this occurs mostly in flat

bones, vertebrae, Iliac bones, Ribs

and the ends of long limbs

Thymus

- Where maturity of T-cells occurs

- Bi- lobed

- Medulla and cortex regions

- No change during immune response to antigens, continuous development of T cells


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- Hassalls corpuscle secretes soluble factors, and is important in regulatory T cells

Secondary Lymphoid Organs

Lymphatic System

- Fluid drained from between tissue cells absorbed into lymph

- 2 to 3 litres of lymph are returned to the blood each day (via superior vena cava)

- In the process of draining, lymph can capture pathogens

- Fluid passes through lymph nodes which survey for pathogens

LYMPH NODES

- Kidney shaped organs > 1cm

- During immune response, swell in size

- Fluid enters through AFFERENT vessel

- Fluid leaves via EFFERENT vessel

- Lymph perculates through all lymphocytes before

leaving the node

- Usually a SUMMATIVE junction, i.e. there are many

afferent vessels but one efferent vessel

- Rich blood supply lets lymphocytes into the lymph

nodes via the HIGH ENDOLTHELIAL VENUES

- T-cell zone: parafollicular cortex

- B-cell zone: lymphoid follicle- mostly on the

periphery of the lymph node

- During immune response, there is a massive proliferation of B cells, which leads to the formation of a

GERMINAL CENTRE

- Specific chemokines target their respective lymphocytes to their specific areas, e.g. T-cells to the

parafollicular cortex

- The lymph entering lymph nodes may also contain cells such as dendritic cells and macrophages

Spleen

- Filter for antigens in the blood

- Large organ in the abdomen

- Separated into

white pulp: lymphoid cells around blood vessels, full

of lymphocytes

red pulp: contains old damaged RBC

- Any diseases involving RBC, i.e. sickle-cell, often

results in an enlargement of the spleen

- T cell area: peri-arteriolar lymphatic sheath (PALS)

- B cell area is located further away from blood vessels

- Not a vital organ: Individuals who do not have a spleen are highly susceptible to infections with encapsulated

bacteria

Mucosal Associated Lymphoid Tissue (MALT)

Epithelium is the first line of defence mucosae and skin form a physical barrier very large surface area, in large part a single layer of cells heavily defended by the immune system in case it breaks


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Gut Associated Lymphoid Tissue

- Many villi, plus smoother regions - Involved in the mesenteric lymphatic drainage

system to mesenteric lymph nodes, including intraepithelial lymphocytes

- PEYERS PATCH: non-capsulated aggregation of lymphoid tissue- predominantly B lymphocytes and contain germinal centres during immune responses

- M-CELLS: sample contents of the intestine,

surveying for pathogens which they can then deliver to immune cells

Cutaneous Immune System - I.e. the skin - Epidermis contains keratinocytes, Langerhans cells

and intraepidermal lymphocytes - The dermis heavily guards the epidermis with

immune cells, e.g. macrophages, T lymphocytes etc - The demis also consists of venules and lymphatic

vessels, providing entry to the blood circulation and drainage to regional lymph node

3. Outline the recirculation of lymphocytes. PROBLEM: There are a very large number of T cells with different specificities There are a very large number of B cells with different specificities There may only be limited amounts of antigen How does the body ensure that the antigen meets lymphocyte with specific receptor? SOLUTION:

Lymphocyte recirculation - Pathogen on mucosal surface - Naive lymphocytes leave BM and Thymus and enter the bloodstream - Recirculate through peripheral lymphoid tissue - Recognition of antigen- massive B cell proliferation in secondary lymphoid tissue (lymphocyte activation) - Otherwise the lympcytes die

Extravasion of naive T cells into the lymph nodes (occurs during immune response)

- The naive T cell rolls along the epithelium

- These are then stopped and activated by specific chemokines at a particular place on the epithelium. This right place is determined by SELECTINS


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- INTEGRINS then increase adhesion of the T cell to the epithelium, leading to arrest of the cell - Transendothelial migration of the T cell from the bloodstream into the lymph node then occurs - Antigens also enter the lymph nodes via the draining lymphatics - Naive lymphocytes recirculate approx once per day -- enter lymph nodehigh endothelial venue

lymphocyte is activated by antigen stops recirculatng massive proliferation of B lymphocytes reenter the blood via the superior vena cava (via the efferent vessel) target invading microbes/pathogens

Anatomical structure of the immune system

4. Explain the use of CD (cluster of differentiation) markers for discrimination between lymphocytes. Lymphocytes

Small cells with agranular cytoplasm and a large nucleus Can be subdivided into 2 groups depending on where they were produced

- B lymphocytes (Bone Marrow) - T lymphocytes (Thymus)

These express different CD molecules, which are recognised by different antibodies CD Markers

an internationally recognised systematic nomenclature for cell surface molecules used to discriminate between cells of the haematopoietic system more than 300 CD markers clinical importance e.g. CD4 in HIV

5. Compare and contrast phenotypic characteristics of B and T cells. Relative Quantities

T cells B cells

7.5 x 109 in the blood

Blood contains 2% of the total pool, therefore 50 x 7.5 x 109 = 3.75 x 1011

~ 1012, but mostly in the gut

T Lymphocytes

all express CD3- antigen specific receptor (TCR)

TCR, about 10% in blood

TCR, about 90% in blood: ~2/3 express CD4, ~1/3 express CD8. All mature T cells express one or the other

CD4+ = T helper cells, regulatory T cells- Secrete cytokines CD8+ = cytotoxic T cells- Lyse infected cells, secrete cytokines Thymic output of naive T cells declines with age, and the thymus atrophies. Therefore older people have a

reduced ability to respond to new infections. However the total number of T cells does not change, there are just more memory cells. ANTIGEN RECOGNITION

only recognise processed antigen presented at the surface of another cell using T cell receptor antigen is presented by an MHC molecule

B lymphocytes

Produced by and develop in bone marrow Surface antigen receptor (B cell receptor) : immunoglobulin like molecule Express CD markers CD19 & CD20 (not CD3, CD4 or CD8) Express MHC Class II (can present antigen to helper T cells) Effector function is to produce antibodies

ANTIGEN RECOGNITION


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recognise intact antigen free in body fluids (so not presented by another molecule)

Use B cell receptor, a membrane anchored form of antibody linked to signalling subunits

6. Give examples of antigen presenting cells (APCs) and their locations. Antigen presenting cells (APC) cells that can present processed antigen (peptides) to T lymphocytes to initiate an acquired (adaptive) immune response:

Dendritic cells (DC) - Location: Widely spread e.g. Skin & mucosal tissue - Presents to T cells

B lymphocytes - Location: lymphoid tissue - Presents to T cells

Macrophages (activated) - Location: lymphoid tissue - Presents to T cells

Follicular dendritic cells - Location: lymph node follicles - Presents whole antigens to B cells


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3. Innate Immunity Dr Keith Gould ([email protected])

1. Briefly describe the functions of the important phagocytic cells: neutrophils, monocytes/macrophages.

2. Define cytokines and describe their general properties.

3. Define complement, list its major functions, and draw a simple diagram of the complement pathways.

4. Describe a typical inflammatory response to a localised infection involving recruitment of neutrophils, and

phagocytosis and killing of bacteria.

5. Briefly outline the events involved in a systemic acute phase response.

6. Outline the phenotype and functions of natural killer (NK) cells.

Innate Immunity

Present from birth- in built

Not antigen specific, but recognizes pathogen-associated molecular patterns (PAMP)

Not enhanced by second exposure, i.e. no memory (comes directly from lymphocytes)

Uses cellular and humoral components in body fluids

Rapid response, cooperates with and directs adaptive immunity

Phagocytosis

Phagocytic cells can ingest whole microorganisms, insoluble particles, dead host cells, cell debris and

activated clotting factors.

In the first step, there has to be adherence of the material to the cell membrane.

Finger-like projections called pseudopodia engulf the material, and a membrane-bound structure called a

phagosome is formed.

This then fuses with a lysosome to form a phagolysosome, mixing the contents of the lysosome with the

engulfed material.

Lysosomes contain hydrogen peroxide, oxygen free-radicals, and various hydrolytic enzymes which can

digest and break down the engulfed material.

Finally, any waste products are released from the cell.

Phagocytic Cells

Neutrophils

- (POLYMORPHONUCLEAR LEUKOCYTE)


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- 50-70% of leukocytes

- short lived cells, circulate in blood then migrate into tissues; first cells to be recruited to a site of tissue

damage/infection

- ~1011 produced per day in a healthy adult, but this can increase approx ten-fold during infection

Macrophages

- less abundant

- dispersed throughout the tissues

- signal infection by release of soluble mediators

Neutrophils

To fight infection, neutrophils:

1. Migrate to site of infection (Diapedesis and Chemotaxis)

- Neutophil rolls along normal endothelium

- At site of damage/when antigen is presented by macrophage, a change in the nature of the endothelium

occurs

- Integrin activation by chemokines- This leads to a change in adhesion molecules into high affinity state- they

flatten out and undergo migration through endothelium

- Chemotaxis- directed migration along chemokine concentration gradient towards area of high concentration

2. Bind pathogen- Opsonisation

- Coating of pathogen with proteins to facilitate phagocytosis

- Opsonins are molecules that bind to antigens and phagocytes

- Antibody and complement function as opsonins

NEUTROPHIL BINDING TO OPSONINS

Bacterium-antibody complex complement activation Fc receptor on phagocyte binds to antibody, CR receptor

to complement opsonins bound to pathogen signal activation of phagocyte

3. Phagocytose

- Key component of host defence

- May result in pus-filled abscess

- Much more effective after OPSONISATION

4. Kill pathogen

- Neutrophil Killing Mechanisms

OXYGEN-INDEPENDENT OXYGEN-DEPENDENT

Uses enzymes: - Lysozyme - Hydrolytic enzymes

Uses Respiratory burst: Toxic Metabolites - Superoxide anion - Hydrogen perozide - Signlet oxygen - Hydroxyl radical

Uses antimicrobial peptides (defensins) Reactive Nitrogen Intermediates: - Nitric oxide

Phagocyte Deficiency

Associated with infections due to extracellular bacteria and fungi

Bacteria

- Staphylococcus aureas

- Pseudomonas aeruginosa


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- Escherichia coli

Fungi

- Candida albicans

- Aspergillus flavus

Deep skin infections, impaired would healing

Poor response to antibiotics

E.g. chronic granulomas disease

Phagocytes

Monocytes

- Circulate in blood

- Smaller than tissue macrophages

- Precursor to tissue macrophages

Macrophages

- Express pathogen recognition receptors (e.g. toll-like receptors TLR, NOD-like receptors NLR, RIG-I: viral

genomes) for many bacterial constituents

- Bacteria bind to macrophage receptors- initiate a response release of cytokine (soluble mediators SIGNAL

INFECTION)

- Phagocytosis then occurs: Engulf and digest bacteria

Cytokines

Small secreted proteins

Cell-to-cell communication

Generally act locally

Powerful at low concentrations

Short-lived

INTERLEUKINS (IL-x) Between leukocytes approx 35 different types

INTERFERONS (IFN) Anti-viral effects approx 20-25 different types

CHEMOKINES Chemotaxis, movement approx 50 different types

GROWTH FACTORS development of immune system

CYTOTOXIC Tumor necrosis factor (TNF)

Mechanism

Inducing stimulus transcription of gene for soluble protein in cytokine-producing cell cytokine binds to

receptor on target cell -- Binding generates signal changes in gene transcription and gene activation

biological effect

Cytokines are usually released in a mixture, therefore have a wide range of effects on a range of different

target cells


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Autocrine Action

same cell

e.g. Interleukin 2

Paracrine Action

nearby cell

e.g. interferon

Endocrine action

circulate in bloodstream distant cell

e.g. interleukin 6

Important Cytokines

IL-1

alarm cytokine

fever

TNF-

alarm cytokine

IL-6

acute phase proteins

liver

IL-8

chemotactic for neutrophils

IL-12

directs adaptive immunity

activates NK cells

Bacterial Septic Shock

Systemic infection

Bacterial endotoxins cause massive release of the TNF- and IL-1 by activated macrophages

Increased vascular permeability

Sever drop in blood pressure

10% mortality

Dendritic Cells

Network of cells located at likely sites of infection, in the skin and near mucosal epithelia

Recognise microbial patterns, secrete cytokines

engulf pathogens, and migrate to local lymph node to present antigens to adaptive immune system

Complement

describe the activity in serum which could complement the ability of specific antibody to cause lysis of bacteria

Ehrlich (1854-1915)

major role in innate and antibody-mediated immunity

complex series of ~30 proteins and glycoproteins, total serum conc. 3-4 mg/ml

triggered enzyme cascade system; initially inactive precursor enzymes, and as a few enzymes are activated,

they catalyse the cleaving of secondary components etc

rapid, highly amplified response

very sensitive

components produced mainly in the liver, but also by monocytes and macrophages


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Activation

The Classical Pathway

initiated by antigen-antibody complexes

The Alternative Pathway

direct activation by pathogen surfaces

The Lectin Pathway

antibody-independent activation of Classical Pathway by lectins which bind to carbohydrates only found on

pathogens, e.g. MBL and CRP

Classical & Alternative Pathways converge at C3

C3 leads to the final Common Pathway

late phase of complement activation

Ends with the formation of the Membrane Attack Complex (MAC)

As a bi-product of the classical pathway, fragments cleaved are

pro-inflammatory molecules

Principle opsonin is C3b

Control Mechanisms

Acheieved by: Lability of components, i.e. their short half-life

Dilution of components in biological fluids

Specific regulatory proteins:

- Circulating/soluble, eg C1-inhibitor, Factor I, Factor H, C4-binding protein

- membrane bound, eg CD59 (interferes with MAC insertion) and DAF (competes for C4b)

Function

1. Lysis

2. Opsonisation

3. Inflammation/chemotaxis

Mast Cells

Secrete histamine and other inflammatory mediators, including cytokines

Mucosal mast cell lung

Connective tissue mast cells skin and peritoneal cavity near blood vessels

Recognise, phagocytose and kill bacteria activated to degranulate by complement products (ANAPHYLATOXINS) leading to vasodilation and increased

vascular permeability. Local Acute Inflammatory Response


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tissue damage trigger cascades:

invasion of pathogens recognition by macrophages phagocytosis release of soluble cytokines + chemokines Diapedesis and Chemotaxis (slowing down of neutrophils in blood vessels and migration towards site of infection)

complement activation mast cell degranulates release of pro-inflammatory fragments + histamines endothelial damage change in nature of endothelium signals site of infection to neutrophils

Systemic Acute-Phase Response

May accompany local inflammatory response 1-2 days after Fever, increased white blood cell production (LEUKOCYTOSIS) Production of acute-phase proteins in the liver Induced by cytokines

ACUTE PHASE PROTEINS Required to enhance immune response

C-reactive protein (CRP) - C polysaccharide of pneumococcus - Activates complement - Levels may increase 1000 fold Mannan Binding Lectin (MBL) - Opsonin for monocytes - Activates complement Complement Fibrinogen - clotting

Importance of Cytokines

Signal liver: - produce acute-phase proteins Signal bone marrow: - Increase Cerebrospinal fluid (CSF) by stromal cells and macrophages - Increase leukocytosis (WBC production) Signal Hypothalamus: - Prostaglandins production fever - Via pituitary gland and adrenal cortex, release corticosteroids signals liver again

Natural Killer (NK) cells

Large granulated lymphocytes

Cytotoxic: lyse target cells ad secrete INTERFERON-

5-10% peripheral blood lymphocytes

No antigen-specific receptor

Complex series of activating and inhibitory receptors

Have receptors which bind to antibody-coated cells (ADCC- ANTIBODY DEPENDENT CELL-MEDIATED

CYTOTOXICITY)

Important in defence against tumour cells and viral infections, especially Herpes

Target Cell Recognition

Missing self recognition

- Ligation of inhibitory NK receptors = inhibition of target cell killing

- Involves recognition of lack of MHC molecules

Induced self recognition

- Ligation of activating NK receptors = target cell killing

- Involves stress-induced molecules


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4. Antibodies Dr Keith Gould ([email protected])

1. Describe with the aid of a simple diagram the immunoglobulin molecule, identifying the antigen-binding site (Fab)

and Fc portions of the molecule.

2. Briefly describe the properties of the antigen-binding site.

3. Distinguish between antibody affinity and avidity.

4. List the immunoglobulin classes and sub-classes in man. Describe their functions and relate these to their

individual structure.

Overview

What is an antibody? A protein that is produced in response to an antigen

Binds specifically to the antigen

Form the class known as IMMUNOGLOBULINS

Large family of soluble GLYCOPROTEINS

Produced by B lymphocytes

Found in serum

>107 different types

Deficiency is life threatening

After binding antigen, initiate secondary effector functions

- Complement activation

- Opsonisation

- Cell activation via specific antibody-binding receptors (Fc receptors)

Structure symmetrical

Two light (25kDa) chains, two heavy (50kDa) chains

Each chain has amino and carboxyl terminal

Chains heald together by disulphide bridges

Electrophoresis of globulins found in serum:

- Relative amounts (decreasing): A, , ,

- Electrophoretic mobility- towards +ve electrode: A, ,

,

Different antibodies therefore have different charges

The discovery of antibody structure Rodney Porter

Limited the digestion of gamma-globulin with purified

papain, which produced 3 fragments in equal amounts

2 fragments had antigen binding activity (Fab)

The third did not, but formed protein crystals (Fc)

Flexibility There is a hinge in the antibody which allows flexibility

between the two Fab


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This allows the angle between the two antigen binding sites to change

angle depending on the proximity of cell surface determinants, i.e.

how close together antigens are

Note: Both light and heavy chains can be divided into variable (where the

sequences are different) and constant (same sequence) regions

Each IG (immunoglobulin/antibody) domain, e.g. variable light, has

INTRAMOLECULAR DISULPHIDE BONDS to maintain their specific 3D

structure required for antigen binding

Many cell surface proteins also have IG-like domains, and are said to belong to the IG super family

The constant region binds to Fc receptors, which can lead to cell activation, e.g. NK cells (secondary effector

functions in immune response)

Antigen-binding site

Antigen binding occurs at 3 HYPERVARIABLE regions, known as COMPLEMENTARITY DETERMINING REGIONS

(CDRs)

These have specific residue positron numbers

The region of binding is a large undulating 3D structure (~750A = 10-10m), so is highly specific and there are a

significant number of interactions between the antibody and antigen surface

Forces involved Hydrogen bonds

Ionic bonds

Hydrophobic interactions

Van der Waals interactions

Are non-covalent, therefore are relatively weak. This means that in order to have a HIGH AFFINITY, there can only be

a short distance between the antigen and antibody, highly complementary nature, and a significant number of

interactions.

Antibody Affinity The strength of the total non-covalent interactions between a single antigen binding site and a single epitope on the

antigen.

The affinity association constant K can be calculated:

K varies from 104 to 1011 L/mol

Antibody Avidity The overall strength of multiple interactions between an antibody with multiple binding sites and a complex antigen

with multiple epitopes

This is a better measure of binding capacity in biological systems

Monovalent interactions have a low affinity

Bivalent interactions have a high affinity

Polyvalent interactions have a very high affinity

Cross-Reactivity Antibodies elicited in response to one antigen can also recognise a different antigen, for example:

1. Vaccination with cowpox induces antibodies which are able to recognise smallpox


21

2. ABO blood group antigens are glycoproteins on red blood cells. Antibodies made against microbial agents on

common intestinal bacteria may cross-react with the glycoproteins, which poses a problem for blood

transfusions.

Isotypes and Allotypes Isotypes are antibodies who are present in everybody, with a constant region.

Allotypes are antibodies that contain single amino acid mutations, giving allelic polymorphisms which vary in

the population

Immunoglobulin Classes

Different classes of antibodies differ in the constant regions of their heavy chains

Class IgG IgA IgM IgD IgE

Heavy chain

CH Domains 3 3 4 3 4

Light Chain / / / / /

IgG and IgA have subclasses

Class IgG IgA

Subclass IgG1, IgG2, IgG3, IgG4 IgA1, IgA2

H chain 1, 2, 3, 4 1, 2

IgG IgA IgM

heavy chain most abundant monomer 4 subclasses- variability mainly

located in hinge region and effector function domains

Actively transported across the placenta- protection from mother to newborn

Found in Blood and extracellular fluids

Major activator of classical complement pathway (mainly IgG1 and IgG3)

Subclasses decrease in proportion from 1-4

heavy chain Second most abundant monomer (blood) dimer (secretions) Major secretory

immunoglobulin Protects mucosal surfaces from

bacteria, viruses and protozoa Secretory IgA: joined by J chain

and secretory component. Plasma cell secretes dimeric form without secretory. This bonds to poly-Ig receptor and is endocytosed and secreted into lumen. The poly-Ig receptor is cleaved and becomes the secretory component

The secretory component

protects IgA from being degraded in the lumen, by proteases etc

heavy chain pentameric 5 monomers joined by J chain

(10 x Fab) mainly confined to blood

(80%) first Ig synthesised after

exposure to antigen (primary antibody response)

multiple binding sites compensate for low affinity

efficient at agglutination of bacteria

activates complement

IgD IgE

heavy chain extremely low serum concentrations least well characterised surface IgD expressed early in B cell

development involved in B cell development and activation

heavy chain present at extremely low levels produced in response to parasitic infections and

in allergic diseases binds to high affinity Fc receptors of mast cells

and basophils


22

cross-linking by antigen triggers mast cell activation and histamine release

Selective Immunoglobulin Distribution

IgG and IgM in blood

IgG in extracellular fluid

Dimeric IgA in secretions across epithelia, including breast milk

Maternal IgG in foetus via placental transfer

IgE with mast cells below epithelium

Brain devoid of antibodies

Antibody effector functions

Summary

Antibodies: In defence

- targeting of infective organisms

- recruitment of effector mechanisms

- neutralisation of toxins

- removal of antigens

- passive immunity in the new born

In medicine

- levels used in diagnosis and monitoring

- pooled antibodies for passive therapy/protection

In laboratory science

- vast range of diagnostic and research applications

Effector Function Activity Example Antibody Class

Neutralization of toxins Inhibits toxicity Tetanus toxin Mainly IgG

Neutralization of viruses Inhibits infectivity Measles Mainly IgG

Neutralization at body surfaces

Inhibits infectivity of bacteria & viruses

Polio Salmonella

Secretory IgA

Agglutination Ag-Ab complexes/ Lattice formation

Bacteria & RBC IgM, IgG

Opsonization Promotes phagocytosis

Bacteria, fungi IgG

Complement activation Classical Pathway

Ag-Ab complex IgM, IgG

Mast Cell sensitisation & triggering

Expulsion Hypersensitivity

Parasites Pollen

IgE

NK cell Cytotoxicity ADCC

Virus infected cells

Mainly IgG


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5. B Lymphocytes Dr Ingrid Muller ([email protected])

1. Describe the process of stimulation of individual B cells to divide and secrete antibody such as to generate

immunity to a particular antigen (clonal selection)

2. Briefly outline the principles of immunoglobulin (Ig) gene rearrangement in the generation of diversity

3. Outline the differences in antibody production during primary and secondary immune responses

4. Differentiate between monoclonal and polyclonal antibody

Adaptive Immune response

B lymphocytes operate during the adaptive immune response

Develops after encounter of antigem

Takes 4-7 days to develop and become effective

Elicited antibody production specific to encountered antigen

2 types:

Humoral- B cells -- antibodies

Cell Medicated- T cells -- cytokines, lysis of pathogens

B Lymphocytes

White blood cells

Derived from haemopoietic stem cells

Are effector cells of humoral immunity; they secrete antibodies and form memory cells

Where do they come from? Derived in the bone marrow in the absence of antigens

Mature in the bone marrow, whereby they express specific B cell receptors (BCR)

Migrate into the circulation (blood, lymphatic system) and into lymphoid tissues

Antibody production requires antigen-induced B cell activation and differentiation- this occurs in peripheral

lymphoid organs

B cell Maturation Pro-B Cell Pre-B Cell Immature B Cell Mature B Cell

Occurs in the bone marrow in the absence of antigen

Mature B cells are specific for a particular antigen- their specificity

resides in B cell receptor (BCR); a membrane bound immunoglobulin

B cell Receptor (BCR) Transmembrane protein complex composed of:

mIg

- central larger immunoglobulin molecule

- cytoplasmic tail too short so is not involved in signalling

Ig/Ig

- di-sulfate linked heterodimers

- contain immunoglobulin-fold structure

- cytoplasmic tails of Ig/Ig is long enough to interact with intracellular

signalling molecules

has a unique binding site- binds to ANTIGENIC DETERMINANT or

EPITOPE -made before the cell ever encounters antigen

large monoclonal population on surface of the B lymphocyte


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Antigen and BCR diversity For the immune system to respond to the large number of antigens we are exposed to, we need to have a

large REPERTOIRE of specific BCR on different B cells that can recognise the huge array of antigens

1010 different antibody molecules can be generated by B cells with specific BCR

Functional BCR genes do not exist until they are generated during lymphocyte development

Each BCR chain ( & light chains, and heavy chain) is encoded by separate MULTIGENE FAMILIES ON

DIFFERENT CHROMOSOMES

During maturation, these gene segments are rearranged and brought together to form the BCR

IMMUNOGLOBULIN GENE REARRANGEMENT

There are a number of VARIABLE; V, DIVERSITY;D and JOINING;J gene segments that may be responsible for

each chain. The Diversity segment is only associated with the heavy chain. There is also a CONSTANT REGION

associated with each chain

This generates the diversity of the lymphocyte repertoire

Prototypical Membrane Protein Synthesis Genomic DNA (transcription) Primary transcript RNA/pre-mRNA (Splicing) Mature mRNA

(translation) Membrane protein

Intracellular; Amino terminus of protein and protein domains relating to specific exons

Transmembrane; relates to specific exon/s

Extracellular; cytoplasmic tail- consists of exons and carboxyl terminus

Light Chain Synthesis Germline DNA (rearrangement of V and J segments involving VDJ RECOMBINASE) B cell DNA

(Transcription) Primary transcript RNA/pre-mRNA (Splicing) Mature mRNA (translation) Light chain

polypeptide (Kappa or Lamda)

During joining of gene segments the unused DNA is looped out and removed (Germline DNA B cell DNA)

Heavy Chain Synthesis Germline DNA (rearrangement of V and J segments involving VDJ RECOMBINASE) B cell DNA

(Transcription) Primary transcript RNA/pre-mRNA (Alternative Splicing) Mature mRNA (translation)

Heavy chain polypeptide

ALTERNATIVE SPLICING; results in different mature mRNA, as the mRNA express different genes (e.g. they

may have different constant region genes present)

BCR rearrangement Required for B cell maturation

Adaptive Immune Response

Antibody production is a highly regulated process after activation by epitope

If a B cell does not meet an antigen death


25

Antibodies may keep specificity but change

class

During immune response, the first antibody

produced is IgM, but this can change

The adaptive immune response is characterised

by:

1. Specificity

2. Diversity

3. Memory

Clonal Selection Basis of adaptive immunity

Non-self reactive mature lymphocytes then

migrate to the periphery

Our immune system is usually exposed to multiple antigens, therefore multiple cells will be activated

Each lymphocyte (T or B) expresses an antigen receptor with a unique specificity,

Binding of antigen to its specific receptor leads to activation of the cell, causing it to proliferate into a clone

of cells

All of these clonally expanded cells bear receptors of the same specificity to the parental cell

Lymphocytes expressing receptors that recognize self molecules are deleted early during lymphocyte

development and are phagocytosed/lysed

Result: Plasma Cells, Antibodies, Memory cells

Antibody production Naive antigen-specific lymphocytes cannot be activated by antigen alone; they require accessory signals

either from:

- Microbial Constituents- Thymus Independent

- Helper T cells- Thymus Dependent

Thymus Independent Thymus Dependent

- Microbial Consistuents - Only IgM is produced - No memory cells formed - Antigens directly activate B cells without the

help of T cells - This can induce antibodies in people with no

thymus and no T cells (Di-George syndrome) - The second signal required is either

provided by the microbial constituent or by an accessory cell

- Helper T cells - All Ig-classes produced - Memory is formed - Membrane bound BCR binds with antigen

and is internalised and delivered to intracellular sites

- Antigen is degraded into peptides - Peptides associated with Self- MHC Class II,

forming a complex which is expressed at the cell surface

- T lymphocytes with a complementary T cell receptor (TCR) recognises the complex

- T helper cells then secrete LYMPHOKINES - B cell then enters the cell cycle, forming a

clone of cells with identical BCRs- differentiating into plasma and memory cells

T-B cell collaboration Antigen cross link with BCR induces signal 1-- MHC II, B7

Antigen is internalised and degraded, and the peptide-MHC II complex is presented


26

T cell recognises complex and co-stimulation by B7and CD28 interaction activation of T cells

B7(expressed by B cell)

CD28(expressed by TH cell)

Activated T cell expresses CD40L

The interaction between CD40L and CD40 (expressed by B cell) induces signal 2

Activated B cells (CENTROBLAST) express cytokine receptors

T cell derived cytokines bind to receptors on B cells

B cells proliferate and differentiate into antibody secreting plasma cells

Cytokines

Certain cytokines help to produce certain Ig classes during differentiation of CENTROCYTES into plasma cells

Class switching During class switching, the variable region (and hence the specificity) remains constant

However the constant region changes from the original IgM


27

Example of Ig class switching above

Immunological Memory

Consequence of clonal selection and antigen recognition

Memory responses are characterised by a more rapid, heightened and more efficient immune reaction that

serves to eliminate pathogens fast and prevent diseases

Can confer life-long immunity

Initial antigen contact induces a PRIMARY RESPONSE

Subsequent encounter with the same antigen will induce a SECONDARY RESPONSE which is more rapid and

higher

The secondary response reflects the activity of the clonally expanded population of MEMORY B CELLS

The primary response consists of mainly IgM, whereas the secondary response will involve other Ig classes

Immunological memory forms the basis for immunisation

B cell memory: Increase in antibody amount and antigen affinity

Property Primary Response Secondary Response

Responding B cell Naive Memory

Lag period 4-7 days 1-3 days

Time of peak response 7-10 days 3-5 days

Magnitude of peak antibody response

Varies depending on antigen 100-1000x greater

Isotype produced Predominantly IgM Predominantly IgG

Antigens Thymus independent and thymus dependent

Thymus dependent

Antibody affinity Lower higher

Polyclonal and Monoclonal antibodies

Polyclonal antiserum- all antigenic epitopes induce an immune response many different B cells activated

different antibodies produced

Invading microorganisms have multiple antigenic epitopes A mixture of antibodies directed to several

antigenic determinants will be produced which are derived from many different clones of B cells = polyclonal

response

Monoclonal antibodies are derived from a single B cell clone, which can be extracted after first combining

the plasma cells with myeloma cells to form hybridomas. Monoclonal antibodies are used to quantify CD4

count in HIV patients


28

Myeloma = cancerous plasma cells that divides permanently without antigenic stimulation and secretes

antibodies which are indistinguishable from normal antibody = myeloma proteins. They confer immortality

when hybridised with another cell

Plasmacytoma - clone of malignant plasma cells


29

6. T lymphocytes and Antigen recognition Dr Keith Gould ([email protected])

1. Outline the origins and functions of T lymphocyte subsets.

2. Briefly describe the structure and distribution of major histocompatibility complex (MHC) class I and class II

molecules.

3. Outline the mechanisms by which antigen presenting cells (APCs) process and present antigens.

4. Compare and contrast antigen recognition by B and T lymphocytes and by CD4+ and CD8+ T lymphocytes

T lymphocytes

Destroy intracellular pathogens

T cell receptor (TCR) recognizes small peptide fragment of antigen presented by MHC molecule on the

surface of host infected cell

T cell receptor (TCR) Analogous to membrane bound Fab portion of antibody

The variable region is towards the N terminus

The constant region is towards the membrane

The cytoplasmic tail is too short for signaling, so the polypeptides associate

with CD3 POLYPEPTIDES with longer CYTOPLASMIC DOMAINS- this is critical

for signaling.

CD3 polypeptides may consist of GAMMA, DELTA, EPSILON and ZETA subsets

Antigen Recognition 2 major populations of T cells:

- CD4+: use CD4 co-receptor, see peptides on MHC class II- class II restricted

- CD8+: use CD8 co-receptor, see peptides on MHC class I- class I restricted

CO-RECEPTOR molecules bind to the relavent MHC, increasing the avidity of T CELL-TARGET CELL

INTERACTION

Important in signalling

Target Cell Destroying CD8 (Tc or CTL)

- most are cytotoxic and kill target cells - also secrete cytokines

- Induce apoptosis in the target cell (programmed cell death, suicide)

CD4 (T helper cells, Th)

- secrete cytokines

- Recruit effector cells of innate immunity

- help activate macrophages

- Amplify and help Tc and B cell responses

MHC molecules present antigen fragments at cell surface

CD8+ CTL- kill target cells, e.g. viruses

CD4+ TH1- activate macrophages

CD4+ TH2- amplify antigen-specific B cell response


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The Thymus

Full of lymphocytes, but no immune response to infection

T cell precursors; PROGENITOR CELLS, develop in the bone marrow and migrate towards the thymus in the

circulation

Maturation of the Thymocytes occurs from the CORTEX to the MEDULLA

Mature THYMOCYTES/T cells then are transported out of the thymus and around the body via the circulation

Development 1. T cells are CD4- and CD8- (they express neither; double negative)

2. In the cortex, the T cells express a TCR precursor (pre TCR; + surrogate TCR)

3. In the medulla, ~1010 different TCRs created by gene rearrangements. The generated TCRs will only

express either CD4 or CD8

Due to these random gene rearrangements, many of the generated T cells will be SELF-REACTIVE,

therefore these must be destroyed

Selection Occurs during interaction with macrophages and dendritic cells within the thymus. Only useful cells leave the thymus.

Pre TCR checkpoint

- Is the new chain functional?

- No: Death by APOPTOSIS

- Yes: Survival and development to CD4+ CD8+ TCR+

Post TCR checkpoint

- Is the TCR functional?

- Is the TCR dangerous/autoreactive?

- Useless: cannot see MHC die by apoptosis

- Dangerous: see self, i.e. host molecules receive signal to die by apoptosis, i.e. NEGATIVE SELECTION

- Useful: binds weakly to MHC molecule receive signal to survive, i.e. POSITIVE SELECTION

- Note: only 5% of thymocytes survive selection


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Major Histocompatibility Complex (MHC)

Discovery Tumour propagation in mice, i.e. tissue transplants

-acceptance = HISTOCOMPATIBLE

- rejection = HISTOINCOMPATIBLE

Inbred mouse strains - all genes are identical

Transplantation of skin between strains showed that rejection or acceptance was dependent upon the

genetics of each strain

Skin from an inbred mouse grafted onto the same strain of mouse = acceptance

Skin from an inbred mouse grafted onto a different strain of mouse = rejection

Transplantation antigens: MHC Class I

Gene mapping of the same locus shows second class of MHC molecule. This controls the ability to mount an

antibody response; celled IMMUNE RESPONSE GENE- MHC class II

Overview Group of tightly linked genes important in specific immune

responses

Found in all vertebrates

Present antigens to T lymphocytes

MHC Class I Consists of two NON-COVALENTLY ASSOCIATED

polypeptide chains:

- Heavy; 1, 2 3 these are transmembrane

polypeptides with a peptide binding, immunoglobulin like

and cytoplasmic region

- Light; 2-microglobulin this only consists of an

immunoglobulin like region

1 and 2 are joined by PEPTIDE BINDING GROOVE

CD8 interacts with the alpha-3 domain

MHC Class II Consists of 2 transmembrane polypeptides of equal

length

Each polypeptide (alpha and beta) have two domains

CD4 interacts with the beta-2 domain

Cleft Geometry MHC class I

- accommodate peptides of 8-10 amino acids

- Peptide buried within structure

- Peptides all same length

MHC class II

- accommodate peptides of >13 amino acids

- peptides stick out from MHC molecule

individuals have relatively few MHC, but need to present many peptides, so present SUBSETS of peptides

using BINDING MOTIFS

BINDING POCKET: certain residues (anchor residues) are directly associated with the peptide due to their

specific sequence

Binding pockets are useful in order to predict which peptides will be presented


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Human Leukocyte Antigens (HLA) Human MHC molecules

Base DNA sequence- 3.6 million

128 functional genes, only 40% immune-

related

Gene expression Polygenic: there are several gene loci

Co-dominant: both paternal and maternal MHC expressed

MHC Class I: present in nearly all nucleated cells, and levels may be altered during infection or by cytokines

MHC Class II: normally only on professional APC, and may be regulated by cytokines

Polymorphism Large number of alternative different versions of the same gene within the population- termed an ALLELE

Each group of MHC alleles linked on one chromosome is termed MHC HAPLOTYPE

Different MHC Haplotypes lead to different immune responsiveness

>4200 HLA proteins in human population

Most polymorphic: Class I- HLA B, Class II- HLA DR

In reality MHC alleles are NOT randomly distributed in the population: some alleles are much rarer than

others, and alleles segregate with race.

This poses a problem for tissue transplants tissue typing

Antigen Processing and Presentation

T lymphocytes recognize only processed antigens presented on cell surfaces by MHC molecules

ENDOGENOUS antigen: synthesised within cell (taken to CD8)

EXOGENOUS antigen: synthesised outside the cell, and can be taken up by macrophage etc (taken to CD4)

Antigens in different locations require different responses

Different pathways present antigens from

different locations to different T cell

subsets

Class 1:

- Antigen cleaved by proteasome, taken

into RER by TAP (transporter associated

with antigen presenting)

- Bind with MHC class I

- Shaperones, e.g. calnexin, help protein

folding

- Then trafficked by golgi to surface

Class 2:

- Antigen endocytosed

- Cleaved by proteases

- MHC II migrates into RER- associates with INVARIANT chain

- The MHC II invariant complex is migrated into the golgi in ENDOSOME

- Invariant chain is digested by CLIP (Class II associated invariant chain peptide)

- CLIP is then exchanged for the antigenic peptide, which is then presented at the surface

TAP

CLASS I CLASS II

TRANSPORTER

ASSOCIATED

WITH ANTIGEN

PROCESSING

CLIP

CLASS II

ASSOCIATED

INVARIANT

CHAIN PEPTIDE


33

7. Effector T-lymphocytes Dr Ingrid Muller ([email protected])

1. Outline the importance of antigen presenting cells in the induction of T lymphocyte responses

2. Describe effector functions of T lymphocytes including cell-mediated cytotoxicity, macrophage activation, delayed

type hypersensitivity and T/B lymphocyte cooperation

3. Briefly outline the function of T helper cells in relation to the cytokines they produce

4. Explain the different requirements for activation of naive and memory T lymphocytes

T-cell mediated immunity

Different pathogens require different immune defence strategies (intra/extracellular bacteria, virus,

parasites, worms and fungi)

Detects and eliminates intracellular pathogens

Eliminates altered cells, i.e. tumour cells

Location of antigen determines immune response:

o Phagocytes with ingested microbes microbial antigens in vesicles CD4+ effector T cells (TH 1)

o Infected cell with microbes in cytoplasm CD8+ T cells (CTLs)

CD4+ cytokine secretion macrophage activation killing of ingested microbes (also leads to

inflammation)

CD8+ killing of infected cell

CD4+ produce IFN-, IL-2, TNF-

CD8+ secrete granules

Naive T-lymphocytes

Activated in secondary lymphoid organs

Lymphocytes re-circulate in the blood lymph

lymphoid organs

Enter lymph node through specialized areas in post-

capillary venues called HIGH-ENDOTHELIAL VENUES (HEV)

Advantage: recirculation increases likelihood to encounter

antigen

Only effector cells can enter non-lymphoid tissue (not

naive T cells) as they have to have undergone

differentiation process in response to antigen

Naive T cells migrate through secondary lymphoid organs-

this is mediated by receptors on recirculating cells

Encounter with antigen in secondary lymphoid organs activate naive T cells

The migration of naive and effector/memory T cells differs

Dendritic cells, macrophages and B cells are all professional ANTIGEN PRESENTING CELLS (APC) they all

have MHC Class II molecules and lead to the activation of T cells into effector cells

1. Immature DC take up antigen (INNATE IMMUNITY) in the peripheral tissues

2. Immature DC activated leave tissue migrate to secondary lymphoid tissue

3. In the lymph node, the DC matures expresses high levels of peptide/MHC complexes and COSTIMULATORY

molecules therefore leads to more efficient APC


34

Induction and Effector Phases of CMI

Initial Activation

T cells enter HEV in cortex

T cells monitor for antigens presented by APC:

encounter proliferation and differentiation into

EFFECTOR CELLS

non-encounter leave lymph nodes

Both antigen and costimulation is required for T-cell

activation

Costimulatory molecules e.g. CD28 (requires CD80

or CD86 ligand)

Lack of costimulation unresponsive T cells and

can lead to tolerance in peripheral T cells

After Recognition and Costimulation

Recognition proliferation/differentiation

effector function

T-cells secrete IL-2 and IL-2 receptor (required for

proliferation); DIRECT RESPONSE = AUTOCRINE

ACTION

This leads to the cell activation and multiplication

Effector function: APOPTOSIS- destroy infected target cell

Effector T cells are less dependent on costimulation

IL-2

Resting T-cell: moderate affinity receptor (IL-2R + chains only)

Activated T-cell: high affinity receptor ( + + chains) and secretion

Binding of IL-2 and its receptor signals the T cell to enter the cell, which induces proliferation

DEFINITIONS

Nave T cells: mature recirculating T cells that have not yet encountered antigen

Effector T cells: encountered antigen, proliferated and differentiated into cells that participate in the host defense

Target cells: Cells on which effector T cells act

T-effector cells

CD8: peptide + MHC class I- cytotoxic cells

CD4: Th1 cells- interact with macrophages- phagocytosis intracellular bacteria

Th2 cells interact with antigen-specific cell antibody production

CTLs

Naive T cell = CTLp

CTLp is essential a precursor, and must differentiate before it can kill

CTLp does not express IL-2 receptor

Require helper T cells for activation and proliferation (Th1)


35

When binding to their specific antigenic peptide:self-MHC complexes, TCRs and their associated coreceptors

cluster to the site of cell-cell contact.

Clustering of TCRs then signals a reorientation of the cytoskeleton that POLARIZES the effector cell to focus

the release of effector molecules at the site of contact with the target cell.

CTLs contain lytic granules which contain cytotoxic molecules. In the polarized T cells the secretory

apparatus becomes aligned toward the target cell and the content of the lytic granules is secreted.

The lytic granules induces APOPTOSIS

CTLs can kill multiple targets

Early apoptosis: chromatin becomes condensed

Late apoptosis: nucleus very condensed, mitochondria visible, cell loses much of cytoplasm and membrane

Granules

PERFORIN: polymerises to form pore of Target cell

GRANZYMES: serine proteases, activate apoptosis in cytoplasm

GRANULYSIS: induce apoptosis

Cell Death

Granule Exocytosis Pathway

- Perforin Granzymes Cascades

FAS Pathway

- Interaction expression of Fas ligand

on T cell binding initiates cascades

apoptosis

CTL are re-used after dissociation with target

cell

Cytotoxicity

Apoptosis characterised by

fragmentation of nuclear DNA

CTL store PERFORIN, GRANZYMES, GRANULYSIN

Granules released after target recognition

Also release of soluble mediators that contribute to host defence:

- IFN- ; inhibits viral replication and activates macrophages

- TFN and TNF synergise with IFN-

TH cells

The type of TH activated depends on environment e.g. APC and cytokines

The signals the precursors receive correspond to the type of TH: IL-12 TH1 and IL-4 TH2

TH1 and TH2 correspond to MHC II

TH1 activate macrophages in a very regulated and coordinated manner and are involved in opsonisation and

phagocytosis - involving IFN-

TH2 coordinate mast cell degranulation involving IL-4 and release IL-5 eosinophil activtion


36

TH cells coordinate immune

responses to infections with

intracellular pathogens

Cytokine mediated interactions

are also very important

T Helper subset differentiation,

cytokine profile and effector

functions

SEE POWERPOINT FOR MORE DIAGRAMS

Delayed Type Hypersensitivity (DTH) reaction

Mediated by pre-existing antigen specific T cells, mainly by Inflammatory Th1 cells

CD4+ Th1 cells release inflammatory cytokines that affect blood vessels (TNF-), recruit chemokines and

activate macrophages (IFN-)

Can be protective as well as pathological

Primary role in defence against intracellular pathogens

DTH inducers:

- intracellular parasites (Leishmania)

- intracellular bacteria (Mycobacteria)

- intracellular fungi (candida)

- intracellular viruses (Herpes simplex)

If the source of the antigen is not

eradicated chronic stimulation

granuloma formation

If the antigen is not a microbe, DTH

produces tissue injury without

protection HYPERSENSITIVITY

The DTH response consists of two phases: Sensitization phase

Effector phase


37

Clinical and histological appearance

Tuberculin-type hypersensitivity

Reaction characterised by an area of red firm swelling of the skin

Maximal 48-72 hrs after challenge

Histologically there is a dense dermal infiltrate of leukocytes and macrophages

T-B cell collaboration

Immunoglobulin+ B cells bind specific antigen

Ig-antigen complex is internalised, processed and antigenic peptides are presented on the B cell surface in

context with MHC class II

T helper cells with specific TCR recognise antigen-MHC complex

The T-B interactions trigger expression of CD40 ligand on T cells

CD40 ligand will interact with CD40 expressed by B cells

T cells secrete cytokines and B cells express cytokine receptors

The activated B cell then differentiates into antibody secreting plasma cell

T cell Functions

Recognition of antigenic peptides results in T cell activation and:

1. Clearance of pathogen - antigenic peptide derived from foreign pathogen

Pathological reactions can be caused by T cells

2. Autoimmunity - antigenic peptide derived from self protein

3. Rejection (transplants) - antigenic peptide derived from self protein of transplant donor

T helper cells in relation to their cytokines

Th1 associated functions in cell-mediated immunity Cytokines involved

Macrophage activation DTH reaction

IFN-, TNF- IL-2, IFN-, TNF-, IL-3, GM-CSF


38

Help for CD8 cells Down-regulation of Th2 responses

IL-2 IFN-

Th2 associated functions in humoral immunity Cytokines involved

B cell proliferation B cell differentiation and Ig class switching Down-regulation of Th1 responses

IL-2, IL-4, IL-5 IL-2, IL-4, IL-5, IFN-, TGF- IL-4, TGF-, IL-10

Regulator T Cells

Some T cells differentiate into regulatory cells in the thymus or in peripheral tissue

Regulatory T cells inhibit the activation of naive and effector T cells by CONTACT-DEPENDENT INHIBITION or

by CYTOKINE-MEDIATED INHIBITION

Regulate activation and effector functions of other T cells

Natural; 5-10% in body; from thymus and important in autoimmunity

Down-regulate immune response; both cell-to-cell and cytokine mediated

Antigen specific induced

Immunological Memory

Adaptive immune response in which the immune system remembers subsequent encounters with the same

pathogen

Memory responses are characterised by a faster and stronger immune response that serves to eliminate

pathogens and prevent diseases

Can confer life-long immunity to many infections, and is the basis for successful vaccination

Memory cells show qualitatively different and quantitatively enhances responses upon re-exposure

T cell memory

T cells do not undergo isotype switching or affinity maturation

CD45RA expression allows to differentiate between nave memory cells

Expression of the chemokine receptor CCR7, which controls homing to secondary lymphoid organs, allows a

further subdivision of human memory T cells

CCR7- CD45RA- memory cells = effector memory T cells = TEM

CCR7+CD45RA- memory cells = central memory T cells = TCM

TEM: display immediate effector function

TCM: lack immediate effector function, differentiate into CCR7- effector cells after secondary stimulation


39

Following Leishmania infection

(a) The initial events that trigger

development of the most effective CD4+

T-cell response for controlling Leishmania

infection require a primary infection with

live parasites.

The combined stimulus provided by

activated macrophages and their

interaction with nave CD4+ T cells leads

to the development of an effector T-cell

response.

The effector T cells produce cytokines and interact with macrophages and/or monocytes, increasing their

capacity to present antigen (Ag), activate other T cells and kill intracellular parasites.

During this process, effector T cells leave the node and also home to infection sites. The majority of effector

T cells die and, through a series of poorly defined signals, memory T cells are generated.

(b) At least two populations of memory T cells are generated during the immune response CM T cells (i) and

EM T cells (ii) each of which can be defined by a group of functional characteristics and phenotypic

markers.

It is unclear what the specific signals are that induce the formation and maintenance of each subpopulation,

CM T cells are maintained in the absence of live parasites, whereas EM T cells require the presence of live

parasites.

(c) Following secondary stimulation, CM and EM T cells are poised to respond to the challenge, albeit with key

differences.

EM T cells can home immediately to infected lesion sites and produce effector cytokines, whereas CM T cells

must first pass through a phase of activation and differentiation to generate effector cells.

The importance of this delay on inducing rapid protection, and the extent to which CM T cells are influenced

by the EM T-cell compartment are unclear.

Because the most effective protection is provided when both CM and EM T cells are generated and

maintained, a balance of both subsets is important for maximal protection.


40

8. Host Defence Overview Professor Peter Openshaw ([email protected])

Immunity

Protection from infection

Immune response: reaction to a threat (antigen)

Immune system: cells and molecules leading to protection

Role: to defend against viruses, bacteria, fungi, parasites, i.e. dangerous but not SELF things

Our cells are outnumbered by our bacteria 10:1

Modes of transmission of disease

Respiratory

GI tract

Venereal

Zoonoses (vectors)

Defences

Coughing

Sneezing

Mucus

Cilia

Rapid cell turnover

Antimicrobial peptides produced by phagocytes and epithelial cells

General Surface Defence

Mechanical

- Epithelial tight junctions

- Skin- waterproofed by fatty secretions

- Social conditioning, e.g. wahsing

Chemical

- Fatty acids- skin

- Enzymes: lysozyme (saliva, sweat and tears), pepsin (gut)

- Low pH (stomach, sweat)

- Antibacterial peptides (Paneth cells in intestine)

Microbiological:

- Normal flora compete for nutrients/attachment sites

- Production of antibacterial substances

Overview of the Immune Response

Pre-infectionfirst line

- Avoidance

- Small

- Taste

- Mucus

- Physical barriers


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- Surface environment

Early infectionsecond line

- Phagocytes

- Opsonins

- Some lymphocytes

- Interferons

- Acute phase proteins

- Toll-like receptors

Late infectionspecific

- T cells

- Antibody responses

General Trend: Increase in learning and specificity, decrease in breadth of response

Innate Immune System

Innate Sensing

Stranger Model

- PAMPs (pathogen associated molecular patterns) are recognised by dendritic cells

- DC maturation and migration to lymph node

Danger Model

- Necrotic cell death

- DAMPS (damage associated molecule patterns) released, which bind to receptor on DC

- DC maturation and migration to lymph node

Phagocytes

Cells that engulf invaders

Antigen is destroyed in intracellular vesicles

Includes macrophages, neutrophils


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Antimicrobial Defence Mechanisms

Involves neutrophils, Eosinophils, basophils and mast cells

Toxic oxygen, e.g. superoxide O2, H2O2

Toxic nitrogen oxides, e.g. NO

Enzymes, e.g. lysozyme

Antimicrobial peptides, e.g. defensins

DNA nets

Virus Recognition Pathways

Chemical Signals

Interferons

- TYPE I/III: a/b/l

o activates NK cells

o upregulates MHC, Mx

proteins

o activates RNase L, PKR

o induces anti-viral state

- TYPE II: IFNg

o proinflammatory

o Th1 cytokine

o immune interferon


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Chemokines

Cytokines

Innate Cellular Defences

Natural Killer Cells

- kill host cells that are:

o Infected

o Transformed

o Stressed

- Important in viral

infections.

o Viruses evade NK

cell killing

o NK deficiency

leads to increased

infections

- Important early source of

cytokines

- Shape adaptive immune responses

The acquired Immune System

B cells

There are 1014 potential different antibodies (VDJ combinations)

Each antibody recognises one specific shape/charge combination

Each B cell expresses one unique antibody

Antibody binds antigen

Antibody is membrane bound or secreted

Role of antibodies: neutralisation, opsonisation, complement activation

Antibodies may trigger cell mediated cytotoxicity (ADCC)

T cells

Each T cell expresses one TCR

There are potentially 10^18 different TCRs

Each TCR sees a specific

combination of MHC and

peptide at high affinity

Antigen processing and

presentation

Protection against specific

microbes

Defence against bacteria

Surface defences

(mechanical and chemical)

Antibody opsonisation


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Complement (alternative pathway) causing lysis/opsonisation

Phagocytosis

Release of inflammatory mediators and acute phase proteins (also opsonins) etc.

Fever

Mucosal defences

Mannan binding proteins

Antimicrobial peptides

Enzymes e.g. lysozyme

Mucosal lymphocytes

Secretory IgA

Special antigen sampling

o Waldeyers ring

o Peyers patches

o Dendritic cell networks

Defences against viruses

Surface defences

Interferons

Inflammatory mediators and acute phase proteins/opsonins etc.

NK cells

Antibody, complement, ADCC

T cells

Flu Pathogenesis

Factors that affect severity of infection

RNA sequence

Viral load

Environment

DNA of host

Viral Strategies

block IFN induction

decoy IFN receptors

perturbation of IFN signaling

downregulate ISGs

How infection causes disease

Normal response

- Good immune response

- Appropriate regulation

- Pathogen defeated

Immune defect

- Poor immune response

- Poor control of infection

- High pathogen load

Poor T regulatory cells

- Defective regulation


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- Normal viral load

- Uncontrolled immune response

- Sustained, enhanced response

LECTURERS NOTES

Why do we have an immune system?

It can be argued that the immune system has developed to provide us with a survival advantage against infection,

and that all other functions are a by-product. Our internal and external surfaces are bathed in microbes. We inhale

potentially lethal microbes with every breath that we take, and our cells are outnumbered by our bacteria by 10:1,

which form about 3% of our body mass. The essential challenge of the immune system is to remain indifferent to

non-pathogenic microbes, while responding rapidly and appropriately to the constant microbial onslaught.

The physical and chemical barriers: innate defence

A vital barrier to the entry of pathogens is the so-called wall of death, made up of surface layers of skin that are

dead or dying and constantly being shed. Unless the skin is broken by trauma or biting insects, it is very unlikely that

infection can gain access except through the lung or gut. The lung and gut are organs specialised to provide a large

area of contact with the environment, necessary for gas exchange and absorption of food and water. The mucosal

surfaces turn over at a very fast rate, with all the superficial cells being sloughed within a few hours or days. Any

microbe that attaches to these cells is soon lost along with the dead and dying cells. The mucocilliary system in the

lungs clears microbes from the lung; Cystic fibrosis patients cannot form mucus normally and suffer from recurrent

respiratory infections. Mechanical defence should not be underestimated.

There are also chemicals (fatty acids, enzymes etc.) that bathe the skin and other body surfaces: Lysozyme in our

tears digests bacterial cell walls and the acid in our stomachs kill many of the microbes we ingest. The normal flora

in our gut prevents other bacteria from gaining a foothold, so affecting susceptibility to gut infections following

antibiotic treatment.

Detection of Pathogens by the Innate Immune System

The innate immune system is our first line of defence against infection. Its components are generally innate, i.e. pre-

formed, and rapidly react to pathogen invasion. Classically, the innate system does not adapt and therefore shows

no memory response.

Recognition is based on the sensing of common molecular patterns on the surface of pathogens, a signal that is

contingent on whether or not that particular foreign component is normally present at the site concerned.

Therefore, a molecular pattern may be sensed at the surface and lead to no response, whereas the same molecular

pattern sensed in the cytosol may induce a vigorous reaction. The toll-like receptors (TLR) are an excellent example

of this pathogen sensing system. 204

The complement system is a pre-formed protein cascade which can rapidly punch holes in the outer membrane of

microbes, coat them for phagocytosis (opsonisation) and produces chemoattractants which recruit cellular

components of the immune system.

Chemical signals: interferons, chemokines and cytokines

The production of interferons is also crucial to host defence. Interferons are soluble low molecular weight mediators

released by cells in response to infection, that act both on the cell that releases them (autocrine action) and on other

neighbouring cells (paracrine) to induce an antiviral state and increase defence. The type 1 interferons activate

natural killer (NK) cells and increase the expression of molecules involved in processing and presenting viral proteins


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on the cell surface. The importance of the interferon system to viruses is shown by the very large number of viruses

that have evolved mechanisms to block the synthesis and actions of interferons.

Low molecular weight mediators are also very important in recruiting other cells to the site of information. Cells that

circulate in the blood and lymph migrate out into the tissues in response to infection, particular combinations of

mediators attracting particular cells (by secretion of chemoattractants called chemokines). Neutrophils, for

example, are attracted by a chemokine called interleukin 8 (IL-8). The eosinophils, on the other hand respond to

eotaxin or RANTES.

Cytokines are chemical signals used for communication by the immune system. They may have local and systemic

effects and direct the extent and the nature of the immune response. For example, Interferon-gamma can be

produced by T cells to enhance activation of macrophages. The cytokine TNF-alpha has many systemic effects

associated with infection, including fever and weight loss.

Innate cellular defences

Once in the tissues, the inflammatory cells produce additional chemoattractive or activating mediators, and may

themselves be phagocytic (e.g. they take up particles that are degraded by vesicles within the cells). Macrophages

are important phagocytes which may be tissue resident or be recruited during infection. Neutrophils, which make up

the majority of circulating leukocytes are rapidly recruited to sites of infection. Phagocytes can use their surface

receptors to directly recognise the outer surface of microbes or to recognise other components of the immune

system, including complement and antibody, that have coated or opsonised the microbe surface. The phagocytes

defence mechanisms include toxic enzymes, reactive radicals and defensins that are produced in the phagosome

once the pathogen has been taken up.

Natural killer (NK) cells are regulated by a combination of inhibitory and stimulatory receptors.

Documents

ABS Immunology