Immunology Flashcards
Interleukin 1
Produced by macrophages/basophils
Pro-inflammatory
Macrophages, large granular lymphocytes, B cells, endothelium, fibroblasts, and astrocytes secrete IL-1. T cells, B cells, macrophages, endothelium and tissue cells are the principal targets. IL-1 causes lymphocyte activation, macrophage stimulation, increased leukocyte/endothelial adhesion, fever due to hypothalamus stimulation, and release of acute phase proteins by the liver. It may also cause apoptosis in many cell types and cachexia
Interleukin 2
Produced by T cells
T-, B- and NK cell growth
T cells produce IL-2. The principal targets are T cells. Its primary effects are T-cell proliferation and differentiation, increased cytokine synthesis, potentiating Fas-mediated apoptosis, and promoting regulatory T cell development. It causes proliferation and activation of NK cells and B-cell proliferation and antibody synthesis. Also, it stimulates the activation of cytotoxic lymphocytes and macrophages.
Interleukin 3
T cells and stem cells make IL-3. It functions as a multilineage colony-stimulating factor.
Interleukin 4
Produced by mast cells, T cells and macrophages
Th2 responses
CD4+T cells (Th2) synthesize IL-4, and it acts on both B and T cells. It is a B-cell growth factor and causes IgE and IgG1 isotype selection. It causes Th2 differentiation and proliferation, and it inhibits IFN gamma-mediated activation on macrophages. It promotes mast cell proliferation in vivo.
Interleukin 5
CD4+T cells (Th2) produce IL-5, and its principal targets are B cells. It causes B-cell growth factor and differentiation and IgA selection. Besides, causes eosinophil activation and increased production of these innate immune cells.
Interleukin 6
Produced by Th17 cells and B cells
Pro-inflammatory
T and B lymphocytes, fibroblasts and macrophages make IL-6. B lymphocytes and hepatocytes are its principal targets. IL-6 primary effects include B-cell differentiation and stimulation of acute phase proteins.
Interleukin 7
Produced by bone marrow and thymic stroma
Promote T and NK cell development
Bone marrow stromal cells produce IL-7 that acts on pre-B cells and T cells. It causes B-cell and T-cell proliferation.
Interleukin 8
Monocytes and fibroblasts make IL-8. Its principal targets are neutrophils, basophils, mast cells, macrophages, and keratinocytes. It causes neutrophil chemotaxis, angiogenesis, superoxide release, and granule release.
Interleukin 10
Produced by T-reg cells (also NK and Th2 cells)
Inhibits cytokine production by Th1 cells (anti-inflammatory/dampens immune response)
Th2 cells produce IL-10. Its principal targets are Th1 cells. It causes inhibition of IL-2 and interferon gamma. It decreases the antigen presentation, and MHC class II expression of dendritic cells, co-stimulatory molecules on macrophages and it also downregulates pathogenic Th17 cell responses. It inhibits IL-12 production by macrophages.
Interleukin 12
Produced by dendritic cells, B cells and T cells
Th1 cell differentiation
Monocytes produce IL-12. Its principal targets are T cells. It causes induction of Th1 cells. Besides, it is a potent inducer of interferon gamma production by T lymphocytes and NK cells
TNF-alpha
Produced by phagocytes, lymphocytes, mast cells, etc
Potent mediator of inflammatory response
Interferon gamma
Produced by CD8+ T cells, NK cells
Anti viral response and enhance MHC expression
TGF-beta
Produced by T-reg cells
Anti-inflammatory
MHC Class I
HLA-A, B, C
Present on all nucleated cells
Present endogenous (intracellular) peptides, e.g. tumour, virus, intracellular bacteria
Activate CD8 cells
MHC Class II
HLA-DP, DQ, DR
Present on antigen presenting cells (dendritic cells, macrophages, B-cells)
Present exogenous (extracellular) peptides, e.g. bacteria
Activate CD4 T cells
Th1 cells
Produce Interferon gamma
Immunity against intracellular organisms
Th2 cells
Produce IL-4/5/13
Immunity against helminth
Allergic response
Interleukin 13
CD4+T cells (Th2), NKT cells and mast cells synthesize IL-13. It acts on monocytes, fibroblasts, epithelial cells and B cells. The IL-13 significant effects are B-cell growth and differentiation, stimulates isotype switching to IgE. It causes increased mucus production by epithelial cells, increased collagen synthesis by fibroblasts and inhibits pro-inflammatory cytokine production. Also, IL-13 works together with IL-4 in producing biologic effects associated with allergic inflammation and in defense against parasites.
Th17 cells
Produce IL-17 and IL-22
Immunity against extracellular bacteria and fungi
Tfh cells
Produce IL-21
Required for germinal centre development
Treg cells
Produce IL-10 and TGF-beta
Promotes T cell tolerance
Inhibits T cell activation
Type 1 hypersensitivity
IgE mediated
Mast cell and basophil degranulation
Symptoms: anaphylactic shock, angioedema, urticaria, bronchospasm
Chronology: within 1-6 hours after last intake of allergen
Type 2 hypersensitivity
IgG and complement
Symptoms: Cytopenia
Chronology: 5-15 days after starting allergen
Type 3 hypersensitivity
IgM or IgG and complement or FcR
Deposition of immune complexes
Symptoms: Serum sickness, urticaria, vasculitis
Chronology: 7-8 days (serum sickness/urticaria_ or 7-21 days (vasculitis
Type 4a hypersensitivity
Th1 - interferon gamme
Monocytic inflammation
Symptom: Eczema
Chronology: 1-21 days
Type 4b hypersensitivity
Th2 - interleukin 4 and 5 Eosinophilic inflammation Symptoms: maculopapular exanthem, DRESS Chronology: 1=several days for MPE 2-6 weeks for DRESS
Type 4c hypersensitivity
Cytotoxic T cells (perforin, granzyme B, FasL)
Keratinocyte death mediated by CD4 or CD8 cells
Symptoms: Maculopapular exanthem, SJS/TEN, pustular exanthema
Chronology: 1-2 days for fixed drug eruption
4-28 days for SJS/TEN
Type 4d hypersensitivity
T cells (interleukin 8, CXCL8)
Neutrophilic inflammation
Symptoms: Acute generalised exanthematous pustolisis (AGEP)
Chronology: 1-2 days but could be longer
T cell pathogens
Bacteria: Sepsis
Viruses: CMV, EBV, varicella, resp and intestinal
Fungi/parasite: Candida, P. carinii
B cell pathogens
Bacteria: Strep, staph, H influenzae
Virus: enterovirsus/enteroviral encephalitis
Fungi/parasite: Giardiasis
Granulocyte pathogens
Bacteria: Staph, Pseudomonas, catalase +ve
Virus: N/A
Fungi/parasite: Candida, Aspergillus, Nocardia
Complement
Bacteria: Neisseria, pyogenic bacteria, encapsulated organisms
Viruses: N/A
Fungi/parasite: N/A
Innate immune system - general
i. First line defense
ii. Antigen non-specific, no immunological memory - recognition of molecular patterns (PAMPs) shared by groups of microbes not present in mammalian host eg. lipopolysaccharide (LPS)
iii. Germ-line encoded
iv. Limited diversity
v. Components
1. External barriers
a. Skin and mucous membranes – keratin hostile surface
b. Antimicrobial chemicals
i. Defensins – produced by neutrophils and function to put holes in bacterial membrane and kill them
ii. Lactic acid – inhibits bacterial growth
c. Mucous membranes – urinary, reproductive, GI, respiratory tracts
i. Mucous – traps microbes and cilia move it out
ii. Secretions – flush microbes away (E.g. urine)
iii. Mucous, tears, saliva – contain lysozyme that dissolves cell walls microbes
d. Hyaluronic acid – difficult for microbes to migrate through SC tissue
2. Proteins and bioactive molecules
3. Cellular – phagocytes and NK cells
• First line of defense • Standard response to any attack • Non-specific to antigen • No memory Components 1. Physical and mechanical barriers 2. Proteins and bioactive molecules – complement, cytokines, chemokines 3. Cellular – leukocytes, macrophages and NK cells (viral infection and tumor)
Adaptive immune system - general
i. Antigen specific response
1. Mediated by receptors on cell surface (TCR/BCR)
ii. Generate immunological memory – results in faster, targeted immune response
iii. Components
1. Cellular – T cells (cell mediated) and B cells (humoral)
2. Immunoglobulin
iv. Antigen presenting cells phagocytose Ag and present it to lymphocytes in lymph nodes
• Second line of defense
• Antigen specific response
• Specific rapid response to secondary exposure
• Immune memory
Components
1. Blood proteins – cytokines, chemokines
2. Cellular – T cells (cell-mediated) and B cells (humoral)
Granulocyte cells - list
i. Neutrophils
ii. Eosinophils
iii. Basophils
Antigen presenting cells - list
= able to present antigen to cells of the adaptive immune system (T cells)
i. Dendritic cells
ii. Monocytes
iii. Macrophages
iv. B cells
Lymphohcyte cells - list and gen overview
i. T cells
ii. B cells
iii. NK cells
a. 25% of total WCC in peripheral blood
b. CD34+ HSCT in BM (?)
c. Circulating – T cells 85%, B cells 15%, NK cells 5%
d. IL-2 = T, B and NK cell growth factor
e. IL-7 + IL-15 = T and NK cell development
Phagocytes - general overview
a. Includes
i. Neutrophil
ii. Monocyte
iii. Macrophage
b. Receptors
i. Fc = Ig
ii. C3b receptor = complement
c. Process
i. Attachment – binding of bacteria to phagocyte
1. Non-specific receptors that recognise common pathogens
2. Complement C3 receptor
3. Fc receptor – opsonised antigens bind at Fc receptor site (especially IgG)
ii. Endocytosis
1. Antigen ingested and forms vacuole called phagosome
iii. Digestion
1. Fusion with lysosome that contains hydrolytic enzymes
Lymphoid organs - gen overview
- Primary lymphoid organs
a. Thymus/ bone marrow
b. Develop during first trimester of gestation
c. Thymus = largest at birth, reaches peak mass at puberty then involutes
d. Bone marrow
i. Children, occupies medullary space of almost entire skeleton
ii. Adults, limited to humerus and femur - Secondary lymphoid organs
a. Spleen/ lymph nodes/ tonsils/ Peyer patches/ lamina propria – develop subsequently
b. Peripheral lymphoid tissue ↑ during infancy and childhood, adult size by 6 years of age
e. Lymph nodes
i. Functions = cleanse lymph and site of T and B cell activation
v. Parenchyma divided into:
1. Cortex = where germinal centres form in lymphatic nodules, and B cells differentiate into plasma cells
2. Medulla branched network of lymphocytes, macrophages, reticular cells, plasma cells
f. Tonsils
i. Patches of lymph at entrance to pharynx
ii. Covered by epithelium and have deep tonsillar crypts lined by lymphatic nodules, enclosed by incomplete fibrous capsule
iii. Three groups – pharyngeal (adenoids - nasopharyngeal), lingual, palatine (classic tonsils)
g. Spleen
i. Left hypochondriac region (under ribs 10-12)
ii. Medial hilum = splenic artery/vein, lymphatic vessels
iii. Red pulp
1. Consists of sinuses gorged with erythrocytes
2. Function = produce RBC in fetus and severe anaemia, RBC graveyard as macrophages consume old ones
iv. White pulp
1. Consists of lymphocytes and macrophages along splenic artery
2. Function = antigen surveillance, gets rid of debris/bacteria in blood, regulates plasma volume
Cellular immunity - gen sum
Function = rid of intracellular pathogens – • Viruses • Protozoa/parasites • Intracellular bacteria • Cancer cells • Transplant tissue/cell
Mechanism = cellular response
• Cytotoxic T cells
• Helper T cells assist
• Memory T cells
Activation
• MHCI on all nucleated cells
• MHCII on APCs
Humoral immunity - gen sum
Function = rid of extra-cellular pathogens • Bacteria • Yeasts • Extra-cellular viruses/parasites • Toxins, venoms • Allergens • Mismatch blood transfusion
Mechanism
• B cell and antibody mediated
• Helper T cell assist
• Memory T cells
Activation
• MHCII on APCs
Antigen
o Any molecule that triggers an immune response
o Epitopes – region of an antigen that stimulates the immune response
o One antigen can have several different epitopes that stimulate immunity
o Haptens – too small to be antigens in themselves but can stimulate immune response by binding to host macromolecule and creating epitope
This is often the mechanism for allergy
Immune memory / Ig response
o Memory T and B cells produced in adaptive immunity to initial exposure
o Primary response – initial immune reaction on exposure to antigen for first time
Initial 3-6 day lag
Peak 10 days - IgM
Peak 18 days - IgG
Low within a month
o Secondary response
IgG rises within hours, peaks within few days
Low IgM
No illness
T lymphocytes - general overview
- Key points
a. 25% of total WCC in peripheral blood
b. CD34+ haematopoietic stem cell in the BM (CD = cluster of differentiation)
c. Common lymphoid progenitor
d. Express CD3 and TCR
i. CD3 complex is important for signaling
ii. T cell receptors = recognize antigen in the context of MHC molecules (antigen presenting cells) - 95% alpha-beta
- 5% gamma-delta
e. Development
i. Formed in bone marrow
ii. Mature in thymus = differentiate from CD4+CD8+ to either CD4+ or CD8+
iii. Enter the circulation as naïve T cells and migrate to LNs, spleen and other lymphoid tissue (secondary lymphoid organs) - Development
a. Begin as double negative TCR negative thymocytes (CD4- CD8-)
b. As migrate through the cortex become double positive cells expressing both CD4+ and CD8+ molecules
c. Gene rearrangement of VDJ segments to generate and express a functional T cell receptor
d. Positive selection = recognition of self-HLA molecules by the TCR
i. Class I or class II MHC
ii. Medullary thymus epithelial cells express MHC
iii. If no recognition apoptosis
e. Negative selection = recognition of self-Ag molecules presented in MHC
i. Medullary thymus epithelial cells express MHC with self-antigen from peripheral site
ii. If autoreactive apoptosis
f. If pass positive and negative selection leave thymus as naïve T cell as CD4+ or CD8+ T cell
i. 5% of thymocytes survive positive and negative selection
g. Note that gamma delta T cells are CD4 and CD8 negative (double negative), role poorly understood
h. Process is mediated by AIRE gene – autoimmune regulator
i. Induces the expression of peripheral tissue antigens that are normally only expressed in the periphery
ii. Defects result in APS1 or APECED autoreactive T cells autoimmune disease - VDJ recombination
a. Germline configuration contains variable (V), diversity (D) and joining (J) segments
i. Only beta and delta TCR loci contain diversity segments
b. One V, (D) and J segment are randomly spliced together in a sequentially ordered process
c. Mediated by various enzymes
d. Correlation to disease
i. RAG1/RAG2 mutations -> SCID
ii. Radiosensitivity SCID (eg. DNA Ligase IV, Artemis)
Cytotoxic T cells - general
- Key points
a. CD3+ CD8+
b. Recognition Ag presented in context of HLA class I - Role
a. Immune response to intracellular pathogens (eg. virus) - Two mechanisms
a. Apoptosis via cytotoxicity = release of granules from cytoplasm in the immunological synapse (MHC class I) perforin punches holes in target cell activate caspase cascade within the target cell
b. Binding of FasL to Fas on surface of target cell which activates apoptosis pathway cell death
Helper T cells - general
- Overview
a. All CD4+ T cells are helper T cells and recognize antigen presented in the context of HLA class II
b. Roles depend on specific subtype
i. Enhance T/B cell response
ii. Activate innate immune system
Include: Th1, Th2, Th17, Treg
Th1 cells - general
a. Promote cell mediated response
- summary: activate/differentiate/accumulate macrophages and neutrophils, class switch to IgG, positive feedback on T cells
b. Development
i. IL-12 is secreted by APC and drives the switch from an undifferentiated Th cell to a Th1 cell
ii. IL-12 also activates NK cells, which then secrete IFN-g which can promote differentiation of Th1 cells
iii. IFN-g is also produced by Th1 cells and amplifies the differentiation of Th0 into Th1 cells
iv. Tbet is a transcription factor that is a master regulator for Th1 production
c. Function
i. Activation of macrophages
1. IFN-γ activates macrophages and makes them efficient killers of pathogens
a. Increase production of TNF-α an autocrine signal which induces production of antimicrobial agents such as NO and O2-
b. Increased expression of MHC and costimulatory molecules
c. Secrete IL-12 which acts as a positive feedback loop promoting Th1 differentiation
2. CD40L also interacts with CD30 to activate macrophages
ii. Neutrophil activation = Th1 cells also secrete TNF and lymphotoxins (LT) which can activate neutrophils which also leads to enhanced microbial killing
iii. Class switching = IgG
iv. IL-2 production = induces T cell proliferation, increasing numbers of effector cells
v. IL-3 GM-CSF = induces macrophage differentiation in the bone marrow
vi. CCL2 = Causes macrophages to accumulate at the site of infection
Th2 cells - general
a. Promote humoral response
- summary: activate B cells, class switch IgE, stimulate eosinophils
b. Development
i. IL-4 drives Th2 production; major sources are NKT cells
ii. IL-4 also is “self-amplifying” and promotes differentiation of Th0 cells into Th2
iii. GATA-3 is the transcription factor master regulator for Th2 production
c. Function
i. IL-4 = promoting B cell activation + class switching to IgE
1. IgE binds Fc receptors for IgE on the surface of mast cells
2. When cross-linked, they release cytotoxic granules
ii. IL-5 = mobilises and activates eosinophils IgE, mast cells and eosinophils are all critically important components of the immune defence against helminth
iii. IL-13 = can modify macrophage activation, promote epithelial cell repair and mucous production, promotes smooth muscle contraction → physically expel parasite
Th17 cells - general
- summary: eaerly response to extracellular bacteria and fungi, neutrophil recruitment,
a. Development
i. TGF-β and IL-6 = drives naïve T cells to differentiate into Th17
ii. IL-21 = self-amplifying cytokine
iii. IL-23 = stabilises Th17 phenotype
b. Function
i. These cells secrete IL-17, IL-6 and TNF-alpha
1. IL-17 has an important role in protecting the body from extracellular pathogens eg. Klebsiella pneumoniae, Neisseria gonorrhoeae, Shigella, Staphylococcus aureus and fungal infections
2. May also have a role in anti-tumour immunity
3. Th17 secretes IL-17 at the site of infection which induces pro-inflammatory cytokines + neutrophil recruitment to site of infection
ii. Early immune response to extracellular bacterial infections + fungal infections
1. Increase infiltration of neutrophils
iii. Activate local endothelium
iv. Induce cytokine and chemokine production
v. Autosomal dominant hyperIgE syndrome (STAT3) – no Th17 cells
1. Susceptible to fungal infections
Treg cells - general
Regulatory T cells
- down-modulate immune response
a. Subset of circulating CD4+ T cells that down modulate immune responses
i. Suppress CD4 and CD8 T cells, B cells and NK cells
b. Cell surface expression of CD4 and CD25
c. Nuclear expression of FoxP3 - Transcription factor required for development
d. Development
i. Treg cell differentiation is driven by TGF-β
e. Cytokine production = TGF-beta, IL-10 anti-inflammatory
f. Disease
i. IPEX = deficiency of FoXP3
ii. IL-10/IL-10R defects = early onset IBD
B lymphocytes - background
- Key points
a. BCR (clonally specific) – surface IgM and IgD
b. Express CD19, CD20, CD40, CD79, MHC class II (antigen presenting cell), Fc gamma receptor, C3b receptor (CR1) and CD3d receptor (CR2)
c. Mature in BM, then periphery (secondary lymphoid organs) (antigen independent development = bone marrow)
d. After Ag cross-links Ig, B cells proliferate and differentiate into
i. Plasma cells (in germinal centres of LNs), which lose surface BCR expression and secrete immunoglobulin (antigen-dependent development)
ii. Memory B cell - Development
a. Differentiation from haematopoietic stem cells in bone marrow = antigen independent
b. Mature in peripheral lymphoid organs (eg. spleen, LN) = antigen dependent
c. Cell surface markers alter throughout development - Antigen INDEPENDENT development - VDJ recombination
a. First stage of B cell development is rearrangement of B cell receptor genes
b. Heavy chain VDJ recombination expressed with surrogate light chain survival signals
c. Subsequently light chain VJ recombination
d. If no survival signal will not develop
e. B cell receptor is either IgM or IgD – if successfully rearrange B cell receptor migrate to secondary lymphoid organs
f. Once exposed to antigen undergo clonal expansion plasma cell OR memory cell
g. BTK mutation – X linked agammaglobulinaemia – cannot make a functional B cell - Antigen DEPENDENT development
a. Second phase of B cell development occurs after encounter with antigen in secondary lymphoid organs (eg. lymph nodes and spleen)
b. B cells are activated, proliferate and differentiate into
i. Plasma cell = produce large amounts of Ab of particular antigen specificity
ii. Memory B cell = long-lasting cells able to rapidly produce high-affinity antibodies in response to second antigen challenge
c. Fate of activated B cell depends on antigen presentation and cytokine received – T dependent + independent
T cell dependent B cell response
a. Requires the participation of T helper cells
b. Majority of antibody responses to proteins and glycoproteins
c. B cells located in lymphoid follicle + T cells in parafollicular cortex - interact at the edge of the follicle
d. Process
i. Cross-linking of immunoglobulin receptor (BCR) Ag internalized and processed
ii. Presentation of Ag on surface of B cell to circulating T helper cell
iii. Activation signals from T helper cell
iv. Results in formation of a germinal centre
v. Further proliferation and differentiation into plasma cell
vi. Induction of isotype switching and activation of somatic mutation
e. Isotype switching
i. Only occur in T dependent B cell activation
ii. Naïve B cells express IgM and IgD
iii. T cell derived cytokines induce isotype switching
1. CD40-CD40L interaction (CD40L is on T cells) – stimulates B cells to class switch
2. Mediated by various enyzmes (AID, UNG, APE1, DNA-PK)
iv. Cytokine milieu determines antibody isotype produced
1. IL-10 IgG1/3
2. IL-4/IL-13 IgE
3. TGF-beta IgA
v. VDJ regions (encode for the BCR) are spliced to different heavy chain constant regions – alters the mRNA transcript and encodes a different protein and therefore antibody isotype
f. Somatic hypermutation (SHM)
i. Enables higher affinity Ig reduction
ii. Single base-pair substitutions within the variable region of antibody gene segments
iii. Produce antibody of higher affinity for antigen
iv. Does not alter the antigen specificity
T independent B cell response
a. Some molecules can activate B cells directly - polysaccharides, lipopolysaccharides, polymeric proteins – repeating units that cross-link Ig on B cell surface
b. Advantages
i. Rapid response to pathogens of T cells
c. Limitations
i. Poor induction of memory B cells
ii. Poor affinity maturation of antibody (SHM)
iii. No isotype switching
Immunoglobulins - gen sum
- Key points
a. Produced by the humoral immune system
b. Exquisitely specificity (usually)
c. Range of affinities
d. Low, uM to very high, pM
e. Basis of most vaccines
f. Essential for survival
g. Great diversity of specificities – different 1013 to 1015 potential specificities - Structure
a. Glycoproteins
b. Monomers = pair of identical heavy + pair of light chains bound by disulfide bonds
c. Each light and heavy chain has a variable (V) and constant (C) region
d. Variable region is made from VDJ recombination – heavy (VDJ), light (VJ)
e. Immunoglobulin monomers have two antigen binding arms of identical specificity
i. Fab = antigen binding fragment
ii. Fc = constant fragment which binds to various receptors on the surface of cells (eg. NK cells, macrophages) as well as complement - Fc receptor interaction
- Complement
- Binding to a specific receptor responsible for recirculating Ab
- Antibody function
a. Activate B lymphocytes
b. Acts as opsonins
c. Causes antigen clumping and inactivation of bacterial toxins
d. Activates antibody-dependent cellular activity via NK cells
e. Triggers mast cell degranulation parasite, helminth
f. Activates complement (alternative complement pathway) - Immunoglobulin with age
a. IgG – maternal at birth, reaching adult levels at about 5 years
i. Nadir 3-6months due to passive running out and own kicking in slowly
ii. Often present with Ab deficiencies
b. IgM – reach adult levels at 1 year
c. IgA – reach adult levels at adolescence
IgD - general
Structure + Half life
• Monomer
• Half-life 3 days
• 1% of Ig
Properties
• Not secreted
• Transmembrane protein (receptor) of B cells
Function
• Cell surface receptor
• No effector function
IgM - general
Structure/half life
• Monomer or pentamer (10 binding sites)
• Half-life 5 days
• 10% of Ig
Properties
• Transmembrane protein (receptor) of B cells
• Low affinity and high avidity
• Forms a pentamer when secreted – NOT lost in protein-losing enteropathy
• Synthesis begins at 6 days of life, rises to adult levels at 1y
Function
• Primary immune response – 1st Ab secreted in the adaptive immune response (made rapidly)
• Responsible for blood group reactions
• Fixes complement in its uncomplexed form
• Low in newborn period impaired phagocytosis susceptibility to GN bacteria
IgA - general
Structure/half life • Monomer or dimer (rarely trimer) – 4 binding sites • Dimer – secretory • Half-life 6 days • 10-20% of Ig
Properties
• 2 subclasses- IgA1 and IgA2
• Major antibody in mucosal surfaces eg. gut, lungs
• Highest rate of production but serum concentration < IgG as lost through secretion
Function
• Mucosal protection
• Relatively common immunodeficiency, results in regular gastrointestinal infections
• Passive immunity in newborn
IgG - general
Structure/half life
• Monomer
• Half-life 21-28 days
• 70-75% of antibody pool – most abundant in internal body fluids
Properties
• 4 sub-classes (IgG1-IgG4)
• Major serum antibody – peaks 10-14 days after infection
• B cells receive help from Th cells -> isotype switch to IgG
Function
• Responsible for secondary immune response
• Fixes complement
• Antibody-dependent cellular cytotoxicity (ADCC) – Ab bind Fc receptors on NK cells
• Opsonisation
• Neonatal immunity – crosses placental barrier during pregnancy; maternal Ig depleted by 6-8 m, adult levels not reached until 7-8 y
IgE - general
Structure/half life
• Monomer
• Half-life 2 days
Properties
• Serum levels usually very low
Function
• Specialised to fight helminths
• Causes allergy
• Interacts with mast cells, eosinophils and basophils via Fc receptors – stimulates release of histamine and other mediators
Natural killer (NK) cells
- Key points
a. Derived from common lymphoid progenitor - develop in bone marrow
b. Do not express antigen-specific receptors
i. Recognize antigen via germline encoded receptors for pathogen associated molecular patterns
ii. Inhibited by encounter with self-molecules through inhibitory receptors on sell surface
c. IL-7/IL-15 for development
d. IL-2 for growth
e. CD3-CD16+ or CD3-CD56+
f. Produce cytokines after activation eg. IFN-g
g. Recognition of target cell by
i. Fc receptor binding to antibody on the surface of the target cell
ii. TLR
iii. Lack of MHC class I expression or down-regulation of this identifies as abnormal - Function
a. Kill virally infected cells and tumour cells
i. Direct/ cell mediated cytotoxicity - Release granules that directly kill cells (perforin)
ii. Antibody-dependent cellular toxicity (ADCC) via CD16 - Pathogen with antibody on surface
b. Cytokine production (IFN-g, IL-5, IL-13)
c. Contraction of the adaptive immune response
HLA/MHC
HLA = human leukocyte antigen MHC = major histocompatibility complex
a. HLA class I
i. HLA-A, B, C
ii. All nucleated cells
iii. Present endogenous peptides (intracellular/ cytosolic) eg. tumour, virus, bacteria
iv. Alpha 1 and 2 contact peptide
v. One leg in membrane
vi. Binds peptides 8-10 amino acids long (fit inside groove)
vii. Activate CD8 T cells
viii. Type 1 Bare Lymphocyte Syndrome
b. HLA class II
i. HLA-DP, DQ, DR
ii. APCs
iii. Present exogenous peptides (extracellular) eg. bacteria, killed vaccines
iv. Alpha 1, 2 and Beta 1, 2 peptide chains = BOTH CHAINS POLYMORPHIC
v. Alpha 1 and Beta 1 contact peptide
vi. 2 Transmembrane regions
vii. Binds peptides 13-17 amino acids long (hang outside groove)
viii. Activate CD4 T cells
ix. Type 2 Bare Lymphocyte Syndrome
Antigen presenting cells - general
- Key points
a. MHC/HLA molecules present antigen to T cells
i. Unable to recognise soluble antigen
b. Oligopeptides with antigen-binding groove
c. NO gene rearrangements (unlike BCR/TCR)
d. Variability occurs in the peptide binding region
i. MHC class I = α1 and α2 region
ii. MHC class II = β1 and α1 region
e. Sequence of genes highly conserved = only some differences between humans and mice
h. Codominant expression
i. All alleles expressed in an individual
ii. Each offspring statistically different (4 alleles at each loci – inherit 2)
i. HLA on short arm of chromosome 6 - Antigen presenting cells
a. Also derived from bone marrow precursors
b. Present antigen to T cells
c. Include
i. Dendritic cells
ii. Macrophage + monocyte
iii. B cells
d. Express
i. HLA class I and class II
ii. Accessory molecules (B7 molecules CD80/CD86)
e. Activation of T cell require 2 signals
i. Activation of antigen
ii. Activation signal
f. Without 2 signals become anergic
g. After activation, release cytokines which activate other cells
Lymphocyte activation
a. 2 signals to become activated
i. Antigen
ii. Accessory molecule on surface of
1. APC eg. B7 (CD80/CD86) activation of T cell
a. ICOS deficiency (similar to CD28) type of CVID
2. CD4 T cell eg. CD40 (CD154) activation of B cell
iii. Results in proliferation, cytokine synthesis, effector function
b. If only 1 signal become anergic
Dendritic cells - general
- Sentinels of the immune system
- Relatively rare (0.1% of cells in spleen/lymph node)
• Key features
o Dendritic morphology maximises contact with T cells (single dendritic cell can present antigen to several T cells)
o Short lifespan (3 days)
o Critically important in vaccination
• Function
o Migrate from blood stream to enter skin/ epithelial surfaces
o Internalizes self and foreign antigens
o Present antigen via MHC II
o T cell differentiation
o The only APC that activates naïve T cells and initiate an immune response
• Classification
o Bone marrow derived
Myeloid progenitor
• Produces ‘classical dendritic cells’ – inc Langerhan cells
• Attracted to infection by chemokines
• Present antigen via MHC 2
• Also interact with T cells via CD40/CD40 ligand interaction to stimulate IL-12 production and encourage T cell differentiation
Lymphoid progenitor
• Produces plasmacytoid dendritic cells
o Mesenchyme derived
Follicular dendritic cells – role in activation of B cell
Lack class II MHC
Bind antigen via complement receptors, attract B cells in lymphoid tissues
Cells of the innate immune system - list
- Granulocyte = eosinophil, neutrophil, basophil
* Mononuclear = monocyte, macrophage
Neutrophils - general
- SCF, IL-3, IL-6, IL-11, GM-CSF and GCSF
- Fc gamma receptor
- Arise from bone marrow – arises from granulocyte
- Nucleus with 3-5 lobes
- Neutrophils survive for 6-12 hours in the circulation
- Move to site of infection, phagocytose and kill via oxidative pathway
• Function
o Phagocytose bacteria – mainly intracellular action
o Release antimicrobial chemicals via NADH oxidation pathway – release of highly toxic lysosomal enzymes around cell to kill bacteria (and self)
NADPH oxidase generates large amounts of superoxide (O2-) from molecular oxygen hydrogen peroxide
Myeloperoxidase catalyses reaction of H2O2 to create hypochlorous acid (H-O-Cl)
• Activation process
o Adhere to vascular endothelium via CD18 / L -selectin
This process also involves Siayl Lewis X receptor that binds to e-selectin
o Transmigrate into tissues
o Ingest and kill microbes
o Release chemotactic signals to recruit more neutrophils
Monocytes/Macrophages - general
• CFU-GM monoblast promonocyte monocyte
• Features
o 3-5% WBC
o Structure – kidney shaped nucleus, cytoplasm with small granules
o Arise in BM
o Monocytes in circulation
o Macrophages in tissue (liver/ lungs)
o Express Fc gamma R and complement R1
o Larger than neutrophil
o Cytoplasm filled with granules containing hydrolytic enzymes
o Can survive for weeks – months
• Function
o Phagocytose = receptors for Fc gamma and C3b
o Kill = via oxidative pathways and cytotoxicity
o Stimulate Th cells
Occurs in response to intracellular pathogens
Macrophages release IL12 + TNF alpha
• IL12 stimulates T cells to release IFN gamma
• TNF alpha amplifies macrophage activation
Eosinophils - general
• SCF (stem cell factor), IL-3, IL-5 and GM-CSF (granulocyte-macrophage colony stimulating factor)
• Features
o 2-5% of blood leukocytes
o Non dividing, fully-differentiated cells
o Bilobed nucleus
o Stain reddish brown with eosin
o Contain proteins that are cytotoxic for parasites
o Structure – 2 large nuclei with pink granules in cytoplasm
o Increase in response to parasitic infection, allergy, collagen, spleen/ CNS disease
• Functions
o Phagocytose antigen-antibody complexes, allergens and inflammatory chemicals
o Degranulate and release major basic protein
Implicated in multicellular organisms too large to be phagocytosed
o Secrete proinflammatory cytokines – IL 1,3,4,5, 9 and 13
• Regulation
o Recruited to inflammatory tissues by eotaxin
o Binds to endothelial ligand, marginate between tight junctions of endothelial cells
o Major mediators: IL-5, RANTES, monocyte chemotactic protein MCP3, MCP4
Basophils (and mast cells) - general
• Key features
o Origin – granulocyte
o <0.5-1% of WCC
o Structure – violet granules in cytoplasm
o Increases in VZV, DM, myxoeedema, sinusitis, polycythaemia
• Functions
o Degranulate and release heparin, histamine and other chemical mediators
Improves blood flow to tissue
o Fc epipsilon receptor (IgE)
• Mast cells similar function
Cytokines - gen sum
a. Secreted proteins that are important for
i. Growth
ii. Differentiation
iii. Activation
b. Produced by
i. Antigen presenting cells
ii. Phagocytes
iii. T lymphocytes
c. Action
i. Paracrine – act on neighboring cells
ii. Autocrine – act on same cell that releases them
- Cytokine signaling
a. Most cytokines signal through the same receptor process
b. Cytokine receptor on cell surface
c. Binding of cytokine phosphorylation of JAK activation of STAT form dimer and translocate to nucleus activate gene transcription
d. Defects in cytokine signaling causes primary immunodeficiency
i. SCID - Common gamma chain (X-linked)
- JAK3
- IL-7Ra
ii. STAT3 (hyper IgE syndrome)
iii. STAT1 GOF (chronic mucocutaneous candidiasis)
Complement - key points, components
- Key points
a. Important effector component of innate and adaptive immunity
b. >30 plasma and cell surface proteins
i. Sequential activation
c. Unlike immunoglobulins
i. Heat labile
ii. Part of the innate immune system
d. Function
i. Opsonise – complement receptor mediated phagocytosis
ii. Lyse cells – bacteria, tumour cells, allografts
iii. Mediate inflammation – recruit inflammatory cells - Complement components
a. Components are either
i. Activating
ii. Regulatory
b. Regulation
i. C1 inhibitor
ii. Factors H and I and CD46
iii. CD55 and CD59
c. Deficiencies of regulation
i. Hereditary angioedema (C1 esterase inhibitor)
ii. Atypical HUS Factors H and I and CD46
iii. PNH
Complement - activation, function
- Activation pathways
a. Classical
i. Requires antibody (IgG, IgM) activated by Ag-Ab immune complexes
ii. Sequential activation of C1 (C1qrs), C4 and C2 (C1qrs complex binds Fc portion of IgG/IgM)
b. Alternative
i. Direct activation of C3
ii. Recognition of microbial components on cell surface
iii. No inhibitory/regulators present on microbial cells
c. Lectin (mannose binding lectin)
i. Requires binding of MBL to mannan (surface sugar) on microbial cell surface
ii. Mannose present on particular pathogens – particularly bacteria
- activates C4 - Function
a. C5a and C3b → phagocytosis
i. C3b opsonizes pathogen and can bind to CR1 on macrophage
ii. C5a binding to its receptor on macrophages stimulates the cell to phagocytose C3b coated bacteria
iii. In the absence of C5a, C3b binding to CR1 is not enough to stimulate phagocytosis
b. C3a, C4a, C5a → inflammation
i. The C5a peptide is a POTENT anaphylotoxin which activates the immune system (major antagonist being produced)
ii. C5a generates chemotactic gradients – lots of C5a is liberated surrounding the point of infection and diffuses out into the circulation → leukocytes follow this signal
iii. Summary - Increase vascular permeability and cell-adhesion molecules
- Increased permeability allows fluid leakage from blood vessels allowing Ig and complement to enter interstitial space
- Increased permeability and adhesion promotes migration of leukocytes
c. Membrane attack complex → lysis
i. Consists of C5b, C6, C7, C8, C9
ii. 10-16 molecules of C9 bind to form a pore in the membrane
iii. Results in osmotic lysis
Neonatal immunity - brief summary
• Immunoglobulin
o IgG actively transported across placenta (term infant concentration = maternal)
o Specificity depends on mother’s exposure and response
o Other immunoglobulins not transferred
o Lack effect against E. Coli
• Complement
o Bactericidal against E. Coli, opsonin in phagocytosis of GBS
o No transplacental passage
o Synthesized from first trimester but concentration and activity of complement components ( in preterms)
• Neutrophils o migration (chemotaxis) o adhesion, aggregation o phagocytosis if stress o oxidative respiratory burst of neonatal neutrophils o Neutropenia risk of sepsis
Toll like receptors
• Expressed in epithelial cells, endothelial cells and APCs
• Transmembrane receptors
• Sense components of microbes (cell wall or membranes of bacteria/fungi) and modified nucleic acids or bacteria/ virus
• IRAK4/MyD88 - impaired TLR
o Susceptible to pneumococcal
Superantigens
- Bacterial toxins eg. S aureus Toxic Shock Syndrome Toxin 1 (TSST-1), S pyogenes
- Bind MHC II and TCR beta chain, providing signal to T cell
- NOT processed and do not interact with MHC II via peptide groove
- No specificity and no memory
- Can activate up to 20% (vs 0.001%) of T cells massive cytokine release
Inflammation - general
• Local response to tissue injury or infection
• Functions
o Limit spread of pathogens and destroy them
o Remove debris and damaged tissue
o Initiate repair
• Cardinal signs o Swelling o Redness o Heat o Pain (due to bradykinin and prostaglandins that stimulate pain receptors)
• Stages
- Vasodilation and vascular permeability
a. Histamine, kinins and leukotreines secreted by basophils, mast cells, damaged cells = vasodilation and increased capillary permeability
b. Increased blood flow leading to leukocytes quickly to area - Endothelial adhesion and leukocyte recruitment
a. Endothelial cells produce selectins which adhere circulating leukocytes and draw them into area of inflammation = extravasation
b. Results in margination of leukocytes (adherence to endothelium)
c. Diapedesis follows where leukocytes go through endothelial wall
d. Emigration when enter tissue fluid where inflammation/injury - Neutrophil recruitment and action
a. Chemotaxis – leukotrines and bradykinin guide neutrophils to site of inflammation
b. Neutrophils undertake phagocytosis - Macrophage migration and clean up
a. Neutrophils secrete cytokines which attract macrophages
b. Arrive at site 8-12hours after injury
c. Engulf and destroy bacteria, damaged host cells, dead neutrophils
d. Act as APCs to trigger specific immunity for next exposure
e. Pus = yellow fluid with dead neutrophils, macrophages, cells and tissue debris - Repair
a. Platelets and endothelial cells secrete platelet-derived growth factor that stimulates fibroblasts to multiple and synthesis collage for repair
Fever - general
• Elevation of body temperature
• Causes – infection, trauma, drug reaction, brain tumor
• Functions to facilitate repair
o Promotes interferon activity
o Elevates BMR to accelerate tissue repair
o Inhibits reproduction of bacteria and viruses
• Physiology
o Neutrophils/macrophages phagocytose bacteria and product pyrogen IL-1
o IL1 acts at anterior hypothalamus to secrete prostaglandin E
o PGE raises hypothalamic set point
Response is feeling “cold” – increase temperature by shivering (increase metabolic rate), vasoconstriction
o Decrease set point once infection cleared
Response is feeling “hot” – decrease temperature by vasodilation, flushing, sweating
Lymphocyte markers
i. C45 = pan-leukocyte
ii. CD3 = T cell
iii. CD3/CD4 = helper
iv. CD3/CD8 = cytotoxic
v. CD19 or CD20 = B cell
vi. CD16 and CD56 positive and CD3 negative = NK cell
Immunology investigations - screening tests
B-CELL DEFICIENCY
IgG, IgM, IgA, and IgE levels
Isohemagglutinin titers
Ab response to vaccine antigens (e.g., tetanus, diphtheria, pneumococci, Haemophilus influenzae)
T-CELL DEFICIENCY
Lymphocyte count
Chest x-ray examination for thymic size*
Delayed skin tests (e.g., Candida, tetanus toxoid)
PHAGOCYTIC DEFICIENCY
WBC count, morphology
Respiratory burst assay
COMPLEMENT DEFICIENCY
CH50 activity
C3 level
C4 level
T cell investigations
- Summary of tests
a. Screening tests
i. Lymphocyte count
ii. CXR (thymic size)
iii. Delayed skin tests
b. Advanced tests
i. T cell subset enumeration
ii. Proliferative responses to mitogens, antigens, allogeneic cells
iii. HLA typing
iv. Chromosome analysis - FBE and lymphocyte subsets
a. CD3 – T cell
b. CD3/CD4 – helper T cell
c. CD3/CD8 – cytotoxic T cells
d. Measurement of naïve T cells = marker of thymic output
i. CD3 and CD4/CD8
v. Soluble CD25 (soluble IL-2RA) – increases in malignancy infection, inflammation, HLH
- Naïve T cells
a. Mature T cells that have migrated from thymus
b. Unique antigen-specific TCR, express CD3 and CD4/8
f. TREC – measure of naïve T cells
i. T cell receptor excision circle formed by excision of DNA segments in the process of TCR gene rearrangement
ii. Can be measured in peripheral blood as a surrogate marker of T cell development and thymic output
iii. Absence of TREC on Guthrie card screen for inadequate thymic output; possible SCID - Hoping to add this to the newborn screening test
B cell investigations
- B cells
a. FBE and lymphocyte subsets
i. B cells (CD19 or CD20)
ii. Normally 8-10% of circulating lymphocytes are B cells
iii. Absent = X linked agammaglobulinaemia
iv. Present = CVID, IgA deficiency, HperIgM
b. Memory B cells (deficiency associated with CVID)
i. CD27+
ii. IgD/M +ve or IgD/M –ve
c. Transitional B cells/ plasmablasts - Immunoglobulins
a. Total immunoglobulins
i. IgG, IgA, IgM (in terms of adult levels: IgM > IgG > IgA)
b. Isohaemagluttinins = antibodies to A + B RBC polysaccharide antigens
i. May be absent in first 2 years of life
ii. ALWAYS absent if child is blood type AB
iii. Assesses capacity to make IgM antibodies
c. Vaccine specific antibodies
i. Tetanus (T dependent B cell response) = protein + polysaccharide
ii. Pneumovax 23 (T independent B cell response) = polysaccharide ONLY - Done in children > 2-3 years of age (Children < 2 do not tend to have lasting response to polysaccharide antigens)
d. Specific antibodies by age
i. Changes with age
1. IgG = maternal at birth, reaching adult levels by 5 years
a. Physiological nadir of IgG production at 6 – transient hypogammaglobulinaemia of infancy
2. IgM = reaches adult levels by 1 year, IgA = reach adult levels at adolescence
4. NOTE: IgG subclasses and IgA deficiencies tend to be over diagnosed
ii. T dependent vs T independent response
1. T independent
a. Polysaccharide antigens directly activate B cells (without costimulation of T cells)
b. Results in IgM production
c. Children < 2 do not tend to have lasting response to polysaccharide antigens
i. Susceptible to PS-encapsulated bacteria
2. T dependent
a. Protein-polysaccharide vaccines: protein is processed and presented to CD4 Th cell.
b. T cell then makes IL-4, costimulates via CD40-CD40 ligand IgG antibody
c. Neonates have this form of immune response
Neutrophil tests
Unclear how significant (in Boast notes)
- Screening
a. FBE – neutrophil count - Oxidative burst
- Advanced tests
a. Adhesion molecule assays (CD11b/ CD 18/selectin ligand)
i. CD18 evaluates adhesion function
ii. Tested if suspect LAD
NK Cell tests
- NK cell degranulation
a. Identifies defects in the granulation process
b. Surface expression of CD107a - Intracellular perforin expression
- NK cell cytotoxicity
a. Increasing ratios of effector: target cells (K562 cells)
Complement tests/deficiency tests
- Key points
a. Complement concentrations
i. C3 and C4 –adult levels by 3 months
b. Complement haemolytic activity
i. Classical pathway – adult activity by 3 months
ii. Alternative pathway – adult activity by 1 year - Testing for complement deficiency
a. Do not screen for complement deficiencies with C3 and C4 only
b. If you are looking for a complement deficiency measure the Classical Pathway Activity (CH50 or THC) and Alternative Pathway Activity (AP50) - Recommended tests
a. C3/C4
i. Both low – suggests classical pathway
ii. Normal C4, low C3 – suggests alternative pathway
b. Classical pathway activation (CH50 or THC)
i. Reliable screen for homozygous deficiency in an integral component of classical pathway
ii. Measures capacity of patient’s serum to lyse sheep erythrocytes coated with Ig
iii. All nine components of classical pathway (C1-C9) are required for normal CH50
iv. Heterozygous deficiency – normal CH50 as the level of a component must be reduced by >50% before the CH50 is altered
c. Alternative pathway activity (AP50)
i. Assesses Factor D, B and Properdin
Interpretation
- classic normal, alternative 0 = properdin, factor B or D deficiency
- classic 0, alternative normal = C1, 2, 4 deficiency
- both 0 = C3, C5-9
Inhibitors (look up diagram)
- C1 esterase inhibitor (C1)
- Factor H, I, CD46 (C3b)
- CD 55 and 59 (MAC)
