Basic Immunology 2014

Stephanie Richards

Sharon Choo

Immune system

• Function • Defense against and elimination of pathogenic

microbes and toxic substances • Avoid responses that produce excessive damage to

self-tissues or eliminate beneficial commensal microbes

• Relies on the recognition of the pathogen or toxic substance as distinct from host cells

• Mechanisms that enable this process • Innate immune response• Adaptive immune response

Immune response

• Innate immunity • First line defence • Antigen non-specific, no immunological memory • Components

• Physical and mechanical barriers • Proteins and bioactive molecules • Cellular – phagocytes and NK cells

• Adaptive immunity • Antigen specific response • Generate immunological memory • Components

• Cellular – T cells (cell-mediated) and B cells (humoral) • Blood proteins

Cells of the Immune System

1. Granulocytes

• Neutrophils *

• Eosinophils

• Basophils

3. Lymphocytes

• T cells

• B cells

• NK cells

2. Antigen Presenting Cells

• Dendritic Cells

• Monocytes *

• Macrophages *

• B cells

4. Other e.g. Mast Cells

* Phagocytes: Fc and C3b

Lymphocytes

• 25% of total WCC in peripheral blood

• Derived from common lymphoid progenitor in BM (CD34+)

• IL2: T, B and NK cell growth factor

• IL7 and IL15: T and NK cell development

(a) T cells

• TCR (clonally specific):• 95% alpha-beta

• 5% gamma-delta

• Mature in thymus, where they rearrange their TCRs and express surface CD3 and TCR. They differentiate from CD4+CD8+ to either CD4+ or CD8+, then enter the circulation and migrate to LNs, spleen & other lymphoid tissue

• CD3+CD4+ or CD3+CD8+ (alphabeta+)

(b) B cells

• BCR (clonally specific): surface IgM and IgD

• Express CD19, CD20, CD40, CD79, MHC Class II, Fc gamma receptor, C3b receptor (CR1) and CD3d receptor (CR2)

• Mature in BM, then periphery

• After Ag crosslinks sIg, B cells proliferate and differentiate into plasma cells (in germinal centres of LNs), which secrete immunoglobulins

(c) NK cells

• Receptors are not clonally expressed and genes for the receptors are not rearranged (innate immunity)

• NOT antigen specific

• IL7/IL15 important for development, IL2 growth

• CD3-CD16+ or CD3-CD56+

• Produce cytokines after activation e.g. IFNg

• Kill virally infected cells and tumor cells:• Direct cytotoxicity

• Ab-dept cellular cytotoxicity (ADCC) via CD16

Measurement of lymphocytes in peripheral blood • Cell surface markers

• Detectable by flow cytometry using fluorescent labelled antibodies

• Key markers• CD45 – pan-leucocyte marker

• CD3 – T cell

• CD3/CD4 – Helper T cell

• CD3/CD8 - Cytotoxic T cell

• CD19 or CD20 – B cell

• CD16 and CD56 and CD3 negative – NK cell

Shearer et al JACI

2003;112(5):973-79

Shearer et al JACI

2003;112(5):973-79

T lymphocytes

• Express CD3 and TCR

• TCR allows recognition of specific Ag

• Positive and negative selection in thymus

• Development of wide repertoire TCR capable of recognising foreign antigen

• Deletion of self reactive TCR

• ~5% thymocytes survive positive and negative selection

T cell receptor

V(D)J recombination

RAG1/RAG2 SCID

Radiosensitivity SCID

(eg. DNA Ligase IV,

Artemis)TREC

V-beta repertoire• Recognition of antigen by T cells is

mediated by the TCR• Composed of an α- and β-chain in majority

of cases (5-10% γ/δ)

• Diversity generated by V(D)J recombination during thymic T cell development • Variable (V) genes are grouped in families

consisting of genes with high sequence homology

• Assessment of TCR diversity by V-beta repertoire • Flow cytometry• PCR

TCR signaling

Cytokine signaling

T cell development: Thymus

T cell Development: Thymus

Central Tolerance: Thymus

Central Tolerance: Thymus

* AIRE mutations

– APECED/APS

Naïve T cells

• Mature T cells that have migrated from the thymus

• Unique antigen-specific TCR

• Express CD3 and CD4/8

• Express CD45RA

• TREC

TREC

T lymphocyte subpopulations

• CD8+• Recognise Ag presented in

context of HLA class I

• CD4+ • Recognise Ag presented in

context of HLA class II

• CD4-CD8-• ~5-10% peripheral T cells

• Ligands poorly defined

* Double negative

T cells – ALPS

T lymphocyte subpopulations• Functional subsets

• Cytotoxic function usually CD8+ T cells

• Helper function usually CD4+ T cells

• Cytokine profiles

• Naïve T lymphocytes can differentiate into T helper 1 cells or T helper 2 cells upon activation

• Th1 - Promote cell mediated immune responses

• Th2 - Promote humoral immune responses

• Both CD4 and CD8 cells can exhibit Th1 or Th2 profiles

Cytotoxic lymphocytes

Helper T cells

Th1 and Th2 Lymphocytes

T-helper cell subsets

Plos Pathogens

2014;10(2):1-15

Th17 cells

• CD4+ T cells that produce:

• IL17

• TNFalpha

• IL6

• Early immune response to extracellular bacterial infections

• Increase infiltration of neutrophils

• Activate local endothelium

• Induce cytokine and chemokine production

• HyperIgE syndrome (STAT3) – no Th17 cells

Pro-inflammatory

Regulatory T cells (Treg)

• Subset of circulating CD4+ T cells that down modulate immune responses • Suppress CD4 and CD8 T cells, B cells and NK

cells

• Cell surface expression of CD4 and CD25 • Nuclear expression of Foxp3

• Transcription factor required for development

• Cytokine production • TGF-β• IL-10

• IPEX – Deficiency of Foxp3

Cytokines

• Secreted proteins that are important for

• Growth

• Differentiation

• Activation

• Produced by

• Antigen presenting cells

• Phagocytes

• T lymphocytes

Cytokines • IL-1

• Produced by macrophages/basophils

• Pro-inflammatory

• IL-2

• Produced by T cells

• T, B and NK cell growth

• IL-4

• Produced by mast cells, T cells and macrophages

• Th2 responses

• IL-6

• Produced by Th17 cells, B cells

• Pro-inflammatory

• IL-7

• Produced by bone marrow and thymic stroma

• Promote T and NK cell development

• IL-10

• Produced by Treg cells (also NK cells, Th2 cells)

• Inhibits cytokine production by Th1

cells (anti-inflammatory)

• IL-12

• Produced by dendritic cells, B cells and T cells

• Th1 cell differentiation

• TNF-alpha

• Produced by phagocytes, lymphocytes, mast cells etc...

• Potent mediator of inflammatory response

• IFN-gamma

• Produced by CD8+ T cells, NK cells

• Antiviral response and enhance MHC expression

• TGF-beta

• Produced by Treg cells

• Anti-inflammatory

B Lymphocytes

• Characterised by • Expression of surface Ig receptors (B cell receptor) • Production of immunoglobulins

• Ig receptors allow recognition of Ag• Wide repertoire of Ag recognition depends upon

Ig receptor diversity• Development

• Differentiate from haematopoietic stem cells in bone marrow

• Antigen independent

• Mature in peripheral lymphoid organs (eg. spleen, lymph node)

• Antigen dependent

• Cell surface markers alter throughout development

B cell development

* BTK deficiency – XL

agammaglobulinaemia

B cell receptor (BCR)

BCR signaling

Antigen dependent B cell maturation • Second phase of B cell development occurs

after encounter with antigen • Two fates of developing B cells

• Plasma cell• Produce large amounts of antibody of particular

antigen specificity

• Memory B cell • Long-lasting cells able to rapidly produce high-affinity

antibody in response to second antigen challenge

• Depends on context of antigen presentation and cytokine stimulus received by the B cell • T cell dependent• T cell independent

T cell dependent B cell response

• Requires the participation of T-helper cells

• Majority of antibody responses to proteins and glycoproteins

• Process • Cross-linking of immunoglobulin

receptor (BCR)

• Ag internalised and processed

• Presentation of Ag on surface of B cell to circulating T-helper cell

• Activation signals from T-helper cell

• Further differentiation into plasma or memory B cells

• Induction of istoype switching and

activation of somatic mutation

T cell dependent B cell response – isotype switching & somatic

mutation• Naïve B cells express IgM and IgD

• As B cells mature under the influence of T-helper cells, T cell derived cytokines induce isotype switching

• Mediated by various enzymes (RAG, AID, UNG, APE1, DNA-PK)

• Enables production of antibodies of different isotypesbut same antigenic specificity

• IgG/A/M

• Cytokine milieu determines antibody isotypeproduced

• IL-10 IgG1/3

• IL-4/IL-13 IgE

• TGF-β IgA

Class switch recombination (CSR)

Somatic hypermutation (SHM)

• Single base-pair substitutions within the variable region of antibody gene segments

• Produces antibody of higher affinity for antigen

• Doesn’t alter the antigen specificity

T cell Independent B cell response • Some molecules can activate

B cells directly• Polysaccharides,

lipopolysaccharides, polymeric proteins

• Rapid response to pathogens independent of T cells

• Limitations • Poor induction memory B cells

• Poor affinity maturation of antibody (SHM)

• No isotype switching

Immunoglobulin Molecule

Antibody function(s)

Immunoglobulin isotypes

Properties of Ig Isotypes

Antibody Concentrations by Age: IgG, IgA and IgM

IgG: Maternal at birth, reaching adult

levels at ~5 years

IgM: Reach adult levels at 1 year

IgA: Reach adult levels at Adolescence

Antibody Concentrations by Age: IgG subclasses

IgG subclasses are

also age-dependent,

especially IgG2

IgG subclass and

IgA deficiencies tend

to be overdiagnosed

Antigen Presentation

• MHC molecules present antigen to T cells

• Oligopeptides within antigen-binding groove

• No gene rearrangement (unlike BCR/TCR)

• HLA Class I

– HLA- A, B, C

– All nucleated cells

– Endogenous peptides

(intracellular) e.g. tumor,

virus, bacteria

– Activate CD8 T cells

– Type 1 Bare Lymph Synd

• HLA Class II

– HLA- DP, DQ, DR

– APCs

– Exogenous peptides

(extracellular) e.g. bacteria,

killed vaccines

– Activate CD4 T cells

– Type 2 Bare Lymph Synd

Antigen Presenting Cells

• Also derive from bone marrow precursors• Present antigen to T cells• Express:

• HLA Class I and Class II• Accessory molecules (B7 molecules i.e.

CD80/CD86)

• After activation, release cytokines which activate other cells

• APCs include • Dendritic cells• Macrophages and monocytes• B cells

HLA Class I – Endogenous antigens

HLA Class II – Exogenous antigens

Structure of HLA Class I and II

Superantigens

• Bacterial toxins e.g. S. aureus Toxic Shock Syndrome Toxin 1 (TSST-1), S. pyogenes.

• Bind MHC II and TCR chain, providing signalling 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% (vs0.0001%) of T cells massive cytokine release

Lymphocyte Activation

2 signals to become activated• Antigen

• Accessory molecule on surface of• APC e.g. B7 (CD80/86): activation of T cell

• CD4 T cell e.g. CD40L (CD154): activation of B cell

Cytokine synthesis

If 1 signal only, cell becomes anergic

Signal transduction:• Ig alphabeta (B cells) = CD79a and CD79b

• CD3 (T cells)

T cell Activation

* ICOS deficiency (similar to CD28) – type of CVID

Second

Signal

B cell Activation

B cell Activation

Second

Signal

* CD40L

deficiency –

X-linked

HyperIgM

Apoptosis – programmed cell death

* Fas/FasL/Caspase 10 -

ALPS

Investigation of T cells

• FBC and Lymphocyte subsets • CD3 – T cell • CD3/CD4 – Helper T cell • CD3/CD8 – Cytotoxic T cell

• Measurement of naïve T cells • CD3 and CD4/CD8• Express CD45RA

• Other T cell subsets• Double negative T cells (CD3+/CD4-/CD8-)• Activated T cells (CD3+/CD25+/HLA-DR+)• Regulatory T cells (CD3+/CD4+/CD25+/Foxp3+)

Investigation of T cells

• Lymphocyte proliferation

• Response to mitogen stimulation (PHA)

• Response to anti-CD3 stimulation

• Response to stimulation with specific antigens

(tetanus and candida)

• Lymphocyte signaling

• Phosphorylation of STAT1/STAT3/STAT5

• Presence of co-stimulatory molecules

• CD40L expression on T cells

Investigation of T cells

Measure Cytokines:

• Intracellular

• Plasma

Quantiferon

IL-12/IFN-γ

Th17

Soluble CD25

(sol IL-2Ra)

Th1 pathway

Investigation of B cells

• FBE and lymphocyte subsets

• B cells (CD19 or CD20)

• Memory B cells

• CD27+

• IgD/M +ve or IgD/M-ve

• Total immunoglobulins

• IgG/IgA/IgM

• Vaccine specific antibodies

• Tetanus (T-dependent B cell response)

• Pneumovax 23 (T-independent B cell response)

Natural Killer (NK) cells

• Derived from common lymphoid progenitor• Develop in the bone marrow

• Do not express antigen-specific receptors• Recognise antigen via germline encoded receptors

for pathogen associated molecular patterns • Inhibited by encounter with self molecules through

inhibitory receptors on cell surface

• Functions • Immune response to viral infected cells and

malignancy • Contraction of the adaptive immune response

• Mechanisms • Cell-mediated cytotoxicity • Cytokine production (IFN-γ, IL-5 and IL-13)• Induction of co-stimulatory molecules

NK cell cytotoxicity

Investigation of NK cell function

• NK cell cytoxicity

• Increasing ratios of effector:target cells (K562 cells)

• NK cell degranulation

• Identifies defects in the degranulationprocess

• Surface expression of CD107a

• Intracellular perforin expression

Allergy Testing

• Allergen-specific IgE:

• in blood (“RAST”)

• via skin prick test (histamine release)

IL4

Allergen specific IgE

Allergen specific IgE

SKIN PRICK TEST

Allergen

Allergen-specific IgE

Mast Cell

Degranulation-histamine release

WHEAL

Skin

Mast Cell Tryptase

• Detectable @ 15 minutes

• Time to peak = 1-2 hours

• Return to baseline ~ 6 hrs (t1/2 = 90 minutes)

• Anaphylaxis and Mastocytosis

• Useful for unexpected severe reactions e.g. intraoperative, idiopathic

Antibody Concentrations by Age: IgE

IgE increases with age:

What is “normal” population?

Is “normal” range increasing?

DDx of Elevated Total IgE

• Atopic disease, especially atopic dermatitis*

• Parasitic infestation*

• Allergic bronchopulmonary aspergillosis*

• PID e.g. WAS, hyper-IgE*

• Hodgkin’s disease

• Churg-Strauss syndrome

• IgE myeloma*

* Associated with total IgE >1000 IU/ml

Eosinophils

• SCF, IL3, IL5 and GM-CSF

• Degranulate and release Major Basic Protein

• Multicellular organisms too large to be phagocytosed (e.g. parasites)

• Allergy

Basophils

• Fc epsilon receptor

• Degranulate and release heparin, histamine and other chemical mediators

• Mast cells similar (more later)

Toll Like Receptors

• Expressed in epithelial cells, endothelial cells and APCs

• Sense components of microbes (cell wall or membrane of bacteria/fungi) and modified nucleic acids of bacteria/virus

IRAK4/MyD88

(a) Neutrophils

• SCF, IL3, IL6, IL11, GM-CSF and G-CSF

• Fc gamma receptor

• Arise from bone marrow

• Move to site of infection, phagocytoseand kill via oxidative pathway

(b) Monocytes and Macrophages

• CFU-GM, Monoblast, Promonocyte, Monocyte

• Express Fc gamma R and Complement R 1

• Phagocytose: receptors for Fc gamma and C3b

• Kill: via oxidative pathways and cytotoxicity

• Arise in bone marrow

• Monocytes in circulation

Endothelium

Tissue space

Lumen of post-

capillary venule

Neutrophil

Site of injuryor infection

Chemokine

Selectin

Glycoproteinreceptor

Glycosamino-glycan

Integrin

Adhesion Molecule

Chemokine Receptor

PCAM-1

Murdoch C, 2000

Neutrophil Function

NitroBlue Tetrazolium (NBT) Test

Normal XL CGD Carrier

CGD

Complement

• >30 plasma and cell surface proteins

• Unlike immunoglobulins:• Heat labile

• Part of the innate immune system

• Function:• Opsonise (Complement Receptor mediated

phagocytosis)

• Lyse cells e.g. bacteria, tumor cells, allografts

• Mediate inflammation (recruit inflammatory cells)

Complement Pathways

• Three pathways

• Classical

• requires antibody (IgG/IgM), activated by ag-ab immune complexes

• activation of C1 (C1qrs complex binds Fcportion of IgG/IgM)

• Alternative

• requires contact with bacterial cell surface

• Lectin (mannose binding lectin)

• requires binding of MBL to mannan on microbial cell surface

All 3 pathways lead to production of C3b...

FcR

CR

Properdin

Complement Components

• The components are either:

• Activating

• Regulatory

• Regulation

• C1 inhibitor

• Factors H and I and CD46

• CD55 and CD59

• Deficiencies of regulation

• HAE

• Atypical HUS

• PNH

Complement

Properdin

Factor H

Factor I

CD46

C1

esterase

inhibitor

Inhibitors in red...

CD55

CD59

Complement by Age

The Classical Pathway matures before the Alternative Pathway

Complement concentrations

C3 and C4: adult levels by ~3 months

Complement hemolytic activity

Classical pathway: adult activity by ~3 months

Alternative pathway: adult activity by ~1 year

Testing for Complement Deficiency• Do NOT screen for complement

deficiencies with C3 and C4 only

• If you are looking for a complement deficiency, measure Classical Pathway Activity (CH50 or THC) and Alternative Pathway Activity (AP50)

• Classical Pathway: sheep rbc + antibody; ELISA

• Alternative Pathway: rabbit rbc + chemical; ELISA

Testing for Complement Deficiency

Classical Pathway 0 and

Alternative Pathway Normal?

Classical Pathway 0 and Alternative Pathway 0?

Classical Pathway Normal

and Alternative Pathway 0?

Properdin

C1, C2 or C4 deficiency Properdin, Factor B or D

C3 or C5-C9 deficiency

Testing for Complement Deficiency• Quantitation

• C3 and C4: easily available

• C1q, C2, C5, C6, C7, C8 and C9

• C1 inhibitor

• Factors H and I

• CD46 and CD55/CD59 expression (flow cytometry)

• C1 inhibitor function