Topic 8: The Immune System and Immunochemistry; The Applications of Immunochemistry

What is the innate immune system? What are its functions

first line of defence against pathogens

crucial systemic response to prevent infection and maintain homeostasis

critical for activation and regulation of adaptive immunity

capacity to develop induced response during infection and create inflammation

Compare and Contrast Humoral and Cellular immunity

Humoral

• Abs secreted by B cells bind to antigens

• Ag-Ab complex phagocytosed by macrophages

Cellular

• intracellular Ag in infected cell degraded and presented by MHC

• recognised by T cell Ag receptor 

• T cell makes cytokines that activate macrophage and cytotoxic T cells to kill infected cells

What cells and molecules are involved in humoral and cellular immune systems? What are the targets and antigens of each?

Humoral

• Igs

• targets: bacterial infections and extracellular viruses

• antigen: soluble and surface proteins

Cellular

• MHC proteins and T cell receptors

• cytotoxic T cells

• helper T cells

• targets: infected cells or parasites

• antigen:protein fragments

Briefly describe the humoral immune response

• Ab mediated immune response

• triggers B cells to dev into plasma cells

• plasma cells secrete large amts of Abs

• primary humoral immune response is usually weak and transiet

Briefly describe the cellular immune response

• cell mediated immunity 

• adaptive 

• mediated by T cells

  • mature when binding to MHC-Ag complex on APC releasing cytokines 

  • T cell also releases cytokines 

  • proliferate into T helper and T cytotoxic cells

What is the function of MHC complexes and how do they work?

Function

• bind peptide fragments derived from pathogens

• display them on cell surface for recognition by appropriate T cells

Presentation

• virus infected cells are killed

• macrophages are activated to kill bacteria living in their intracellular vesicles

• B cells are activated to produce antibodies that eliminate or neutralise extracellular pathogens 

Describe the two classes of MHC

MHC class I

• CD8+ cytotoxic T cells

  • endogenous or intracellular peptides

MHC class II 

• CD4+ helper T cells 

  • exogenous and extracellular peptides

cross presentation 

• exogenous antigens can be presented by MHC I 

Where are each class of MHC located? What is their functions (what do they present) and which cells do they attract?

MHC I

• found on surface of nearly all vertebrae cells

• bind and display peptides from degradation of endogenous peptides

• complexes form recognition targets of T cell receptors of cytotoxic T cells 

MHC II

• found on surface of specialised APCs

• bind and display peptides from degradation of external proteins ingested by cells 

• complexes form recognition targets of T cell receptors of helper T cells

what are the general structural features of MHC

• extracellular

• transmembrane

• intracellular domains

• variable regions on extracellular domain

• variable regions involved in protein binding

Describe the different structures of MHC I and II

MHC I

• very polymorphic

• up to 6 variants in each indiv

• consists of 2 types of chains - heavy alpha chain and beta2 microglobulin chain

• alpha chain = variable region

MHC II

• very polymorphic

• up to 12 variants in each indiv

• consists of alpha and beta chains assembled in ER 

• both chains = variable N-terminus 

Describe the different MHC I and MHC II presentation pathway

MHC I

• expressed by all nucleated cells

• assembled in ER

• 30-70% proteins - immediately degraded after synthesis 

• this process viral peptides to be presented very quickly

MHC II

• binds protein fragments

• MHC II + fragment recognised by T cell receptor 

• Tc cells mature and proliferate by interleukins 

• cytotoxic T cells destroy infected cells

What is an antigen?

• antibody generator

• any substance that may be specifically bound by products of adaptive immune system

• biological molecules or chemicals

• typically contain multiple antigenic determinants

What are epitopes?

epitopes are antigenic sites that determine antigen-binding site for Abs

• chemical groups on surface of antigen that can specifically bind antibody or antigen receptors

• 8-26 AA

• linear or comformational epitopes

  • linear = continuous chain of AA and recognised by primary structure

  • comformational = spatially adjacent, discontinuous AA residues and related to 3D structure of proteins 

Where antibodies bind to epitopes? Epitope sites

• pockets

• grooves

• extended surface 

Describe T cell epitopes: How many AA long,

• 12-20 continuous AA peptide fragments of antigen

• combine with MHC

• T cell epitope-MHC II complex expressed on APC surface

• recognised by bound T cell receptor on Th2 cells to produce IL cytokines

Describe T cell cytokines: IL-4, IL-5, IL-13

IL-4

• prolif and diff of B and T cells and enhance function of NK, eosinophils and mast cells

IL-5

• prolif, diff, recruitment, survival and activity of proinflam eosinophilic granulotyes 

IL-13 

• induces expression of TGF-beta1 in macrophages → induce fibrosis

Describe B cell epitopes

• discontinuous conformational polypeptides with certain 3D structure (90%)

• linear polypeptides (10%)

Describe Natural Ig Antibodies. What is the main isotype and what is their function?

• mainly IgM

  • limited genetic repertoire

  • synthesised by B cells in absence of pathogens

• react against foreign antigens and microbe structures

  • activate complement cascade

• neutralise pathogens and helminth parasites and confer virus resistance

What are the mechanisms of Ab action?

the binding of Abs to Ag to form Ab-Ag complexes is the basis of several antigen disposal mechanisms

• precipitation of soluble antigens: soluble antigens joined in a complex → enhance phagocytosis

• agglutination of foreign cells: joins antigenic sites in microbe in a complex → enhance phagocytosis 

• neutralisation: blocks virus/bacteria antigen-host binding sites → enhance phagocytosis

• complement activation/fixation leading to cell lysis 

Describe phagocytic clearance of Ab-Ag complexes

• Ab-Ag complex is recognised by macrophage Ab receptors

• cells internalised by phagocytosis 

• cells killed and degraded

Describe monoclonal and polyclonal Abs

polyclonal

• produced from antiserum of animal immunised with specific antigen

• produced by B cells that may recognise diff epitopes

• heterogenous

  • stable over broad pH and salt conc

  • low specificity and purity

  • inconsistent and limited production

monoclonal

• produced from single cell line

• produces single antibody

• homogenous

  • susceptible to pH and salt conc changes

  • consistent specificty and high purity

  • isolated from renewable sources 

Describe the nature of Ag-Ab interactions. What bonds are involved?

• lock and key concept

• non-covalent bonds

  • h bonds

  • electrostatic bonds

  • van der waals

  • hydrophobic bonds 

• multiple bonds

• reversible

What factors influence Ag-Ab interactions?

Affinity

• measures strength of interactions between epitope and an antibodies binding site 

• single Ag Ab interaction

Avidity

• gives measure of overall strength of Ag-Ab complex 

• dependent on: 

  • affinity of antibody for epitope

  • valency

  • structural arrangement of parts that interact 

cross reactivity

• occurs if Ab raised against specific Ag has competing high affinity toward a diff antigen 

Ag:Ab ratio

physical form of antigen

specificity

How can antibodies be used as assay reagents?

• as hybridomas - make characterised Abs that bind 3D structure of any antigen or target protein 

• widely used in research, dev and diagnostics

  • ELISA

  • western blot

  • HPLC

  • immunohistochemistry

  • therapeutics 

What are the different types of ELISA? What can ELISA be used to screen for?

specific quantitative assay of proteins and other molecules

• indirect: 1st layer Ag → use two types of Abs

• direct: 1st layer Ag → directly use conjugated Ab

• capture/sandwich: 1st layer Ab → sample → conjugated Ab

• competitive

screen for infectious disease or genetic disease

or test for pregnancy 

What are the MOAs of therapeutic antibodies

Neutralisation/blocking

• Ab binds Ag and prevents Ag from interacting with target cell surface molecule

Antibody dependent cell mediated cytotoxic activity (ADCC)

• adaptive immune response mediated by NK cells thru CD16 receptor that finds Fc portion of IgG and triggers lysis of targeted cells

Complement-dependent cytotoxic activity (CDC)

• initiated by complement protein C1q binding to Fc domain of Ab opsonising target cell that triggers activation of complement cascade and leads to lysis of targeted cells

Describe how therapeutic antibodies can neutralise/block pathogens? provide one example

• block pathophysiological function of their target molecules

• Ab bind to ligand or cell surface receptor and block target signalling pathway

• reduced/absent signalling via ligands or receptors alters cell properties in turmour after treatment 

  • reduced cellular activity 

  • inhibition of prolif

  • activation of pro-apoptotic programs

  • tumour cell resensitisation to cytotoxic agents 

Nivolmab is a human IgG4 that blocks PD-1 of malignant melanoma 

Describe how therapeutic antibodies show ADCC activity? provide one example

• triggered when Fc binding domain of antibody binds to specific cell surface antigen on target cell

• binding recruits and activates immune-effector cells i.e. macrophages and NK cells, to lyse target cells

Rituximab is a humanised IgG1 that targets HER-2 and activates ADCC, CDC, and blocking mechanisms against HER-2 pos breast cancer

Describe how therapeutic antibodies show CDC activity? provide one example

• binding of complement complex to Ag-Ab complex → activates complement cascade

• formation of MAC resulting in cell lysis

Denosumab is a human IgG2 that targets CD20 and activates CDC and blocking mechanisms against CLL

Describe how therapeutic antibodies are used as drug delivery carrier systems? provide one example

• used as drug delivery carriers when conjugated to radioisotopes, toxins, drugs or cytokines

• antibody conjugates have advantages over conventional drugs:

  • can b targed directly to tumour tissues

  • achieve higher local conc in tumour tissues

  • cause less “bystander” dmg to neighboring normal cells

Gemtuzumab is a humanised IgG4 that targets CD33 ADC against leukemia

What is the problem with murine Abs being used therapeutically?

• recognised as foreign by human immune system

• short ½ life = 30-40 hours

• constant region lacks effective function

Describe the process of humanising Abs.

• uses DNA tech to synthesis Abs with: 

  • murine CDR

  • human constant region

-mab

• increase similarity to Ab variants produced naturally in humans

• potentially immunogenic

• humanisation is more necessary if longer term therapy is required

What are some strategies for antibody therapeutics? Naked antibodies vs Antibody conjugates?

Naked antibodies

• inhibit vascularisation 

• receptor antagonist antibody

• ADCC 

• CDC

• bispecific Ab

Ab conjugates

• antibody-directed enzyme prodrug therapy 

• immunocytokine

• radioimmunotherapy 

• immunotoxin antibody-drug