ABBAS 3

Question 1

How does Ags induce immune response in B lymphocytes?

Answer 1

o Ag receptors: membrane-bound antibodies
o Can recognise wide variety of macromolecules
 Protein
 Polysaccharides
 Lipids
 Nucleic acids
 Small chemicals in soluble or cell surface-assoc form

Question 2

How does Ags induce immune response in T lymphocytes?

Answer 2

o Can only recognise peptide fragments of protein Ags

o Peptides must be presented by specialised peptide display molecules on host cells

Question 3

What are the barriers in mounting immune response?

Answer 3

o Low freq of naive lymphocytes specific for any 1 Ag

o Diff kinds of microbes need to be combated by diff types of adaptive immune responses

Question 4

Antigens recognised by T lymphocytes

Answer 4

 Most T cells recognise peptide Ags bound to and displayed by Major Histocompatibility Complex (MHC) molecules of APCs
 Each T cell has a dual specificity: TCR recognises peptide Ag and MHC molecule displaying Ag
 Some T cells recognise lipid and other nonpeptide Ags displayed by nonpolymorphic class I MHC-like molecules
 Naive T cells need to see protein Ags presented by DC to initiate clonal expansion and effector cell differentiation
 Differentiated effector T cells need to see Ags presented by APCs to activate effector fxns of T cells in humoral and cell-mediated immune responses

Question 5

MHC and T cell relationship

Answer 5

 MHC: genetic locus whose products fxn as peptide display molecules of immune system
 MHC restriction: characteristic of T lymphocytes that they recognise a foreign peptide Ag only when it is bound to individual’s MHC molecules

Question 6

Dendritic cells

Answer 6

 Network of DC present in
o Epithelia and subepithelial tissues
o T cell-rich areas of peripheral lymphoid organs
o Other organs (less DC)
 Epidermal DC in skin: Langerhans cells
 Epithelial DC
o Immature due to inefficiency at stimulating T cells
o Express membrane receptors to bind microbes – capture and endocytose microbial Ags
o Soluble microbes can enter DC by pinocytosis

Question 7

What happens when microbes bind to TLRs in DCs

Answer 7

Innate system is stimulated.

Production of inflammatory cytokines TNF adn IL-1 is stimulated

Question 8

What molecules are used to activate immature DC?

Answer 8

TLR signalling

Cytokines

Question 9

What happens to Activated DC after activation?

Answer 9

o Lose adhesiveness for epithelia
o Begin to express surface receptor CCR7 – specific for chemoattracting cytokines (chemokines) produced in T cell zones of lymph nodes
o Chemokines direct DCs to exit epithelium and migrate thru lymphatic vessels to lymph nodes
o DCs mature during migration, from Ag-capturing cells to APCs that can stimulate T cells
o Maturation of DC reflected in
 Increased synthesis and stable expression of MHC
 Production of costimulators (req for full T cell response)

Question 10

Where does DC pick up antigens?

Answer 10

 Soluble Ags in lymph picked up by DCs in lymph node
 Blood-borne Ags picked up by DCs in spleen
 Protein Ags are transported and concentrated in regions of lymph nodes where Ags are most likely to encounter T cells

Question 11

Naive T cells

Answer 11

o Continuously recirculate lymph nodes
o Express CCR7 – promote entry of T cells into T cell zones of lymph nodes where they meet DCs carrying captured Ags

 T cell response to Ags introduced to body within 12-18 hours

Question 12

APCs

Answer 12

o DCs
 Most potent APC for activating T lymphocytes

o Macrophages
 Abundant in all tissue
 Cell-mediated immunity: phagocytose microbes and display Ags of microbes to effector T cells
 Effector T cells then activate macrophages to kill microbes

Question 13

Mediation of antigens

Answer 13

A) B cells ingest protein Ags, then display them to helper T cells within lymphoid tissue

B) All nucleated cells can present antigen derived from microbes in cytoplasm in CTLs

C) DC initiate responses of CD8+ T cells to Ags of intracellular microbes
o Cross-presentation/cross-priming
 Professional APC displays the Ags of another cell
 Activates/primes a naive CD8+ CTL
 Infected cell ingested by APC -> microbial Ags processed -> presented in assoc with MHC molecules
 APC also provides costimulation for T cells

D) DC tt ingest infected cells present microbial Ags to CD4+ helper T cells

E) CD8+ T cells diff into CTLs -> kill infected host cells w/o need for DCs or signals other than Ag recognition

Question 14

MHC

Answer 14

 Membrane proteins on APCs
 Display peptide Ags derived from protein Ags for recognition by Ag-specific T lymphocytes
 Individuals identical at MHC locus (inbred animals, identical twins) -> can accept graft from one another
 Individuals with different MHC loci will reject grafts
 Human Leukocyte Antigens (HLA): human MHC proteins
 MHC locus contains 2 sets of polymorphic genes – class I and class II MHC genes – encode class I and II MHC molecules
 MHC locus also contains nonpolymorphic genes – code for proteins involved in Ag presentation

Question 15

Class I MHC molecule

Answer 15

o α chain noncovalently attached to β2-microglobulin (protein encoded by gene outside MHC)
o Peptide-binding cleft/groove
 Formed by amino-terminal α1 and α2 domains
 Peptides 8-11 AA long
 Floor of cleft binds peptides for display to T cells
 Sides and tops of cleft come into contact with TCR
o Polymorphic residue – AA that differ among diff individual’s MHC molecules
 Located in α1 and α2 domains of α chain
 Contribute to variations in floor of cleft – influence ability of diff MHC molecules to bind peptides
 Contribute to variations in tops of clefts – influence recognition by T cells
o α3 domain
 invariant
 contains binding site for T cell co-receptor CD8
o T cell activation needs recognition of MHC-assoc peptide Ag by TCR and simultaneous recognition of MHC by co-receptor
o Thus CD8+ T cells can only respond to peptides displayed by class I MHC (MHC molecules to which CD8 co-receptor binds to)

Question 16

Class II MHC

Answer 16

o Consists of 2 chains – α and β
o Amino-terminal regions of both chains – α1 and β1 domains
 Contain polymorphic residues
 Form cleft tt can accommodate peptides of 10-30 residues
o Β2 domain
 Nonpolymorphic
 Contains binding site for T cell coreceptor CD4
 CD4 binds to class II MHC
 Thus CD4+ T cells can only respond to peptides presented by class II MHC molecules

Question 17

Class I vs Class II MHC

Answer 17

o Mb proteins
o Each contain a peptide-binding cleft at amino-terminal end
o Differ in subunit composition but similar in overall structure

Question 18

Features of MHC - MHC genes are codominantly expressed

Answer 18

o Alleles inherited from both parents are expressed equally

o Class I
 3 polymorphic class I genes (HLA-A, HLA-B, HLA-C)
 Each person inherits 1 set of genes from each parent
 Any cell can express 6 diff class I molecules

o Class II
 Each person inherits 1 pair of HLA-DP, 1 pair of HLA-DQ, and 1 or 2 of HLA-DR
 Heterozygous individual can inherit 6 or 8 class II MHC alleles, 3 or 4 from each parent
 Number of expressed class II molecules can be more than 6 due to extra DR genes

o MHC haplotype
 Set of MHC alleles present on each chromosome
 All heterozygous individuals have 2 HLA haplotypes, 1 from each chromosome

Question 19

MHC genes are highly polymorphic - Features of MHC

Answer 19

o Diff alleles are present among diff individuals in population
o Ensures tt a population can deal with diversity of microbes
o Variations in MHC molecules (thus polymorphism) result from inheritance of distinct DNA sequences, not induced by gene recombination (as they are in Ag receptors)

Question 20

Expression of MHC - Features of MHC

Answer 20

 Class I MHC expressed on all nucleated cells (ie. All cells except RBC)
 Class II MHC expressed mainly on DCs, macrophages and B lymphocytes. Also expressed on thymic epithelial cells and endothelial cells. Can be induced on other cell types by cytokine interferon-γ

Question 21

Peptide-binding cleft of MHC - Features of MHC

Answer 21

o Bind peptides derived from protein Ags

o Display these peptides for recognition by T cells

o Pockets in the floors of clefts
 Place where side chains of AA in peptide Ags fit
 Side chains (anchor residues) in pockets anchor peptides in cleft
 Peptides anchored in cleft by anchor residues contain some residues tt bow upward and are recognised by Ag receptors of T cells

Question 22

Features of interaction of peptides Ags with MHC

Answer 22

1. Each MHC molecule can present only 1 peptide at a time since there is only 1 cleft
2. Each molecule can present many different peptides – MHC has broad specificity for peptide binding
3. MHC molecules only bind peptides
4. MHC acquire peptide cargo during biosynthesis and assembly inside cells
5. Only peptide-loaded MHC molecules are stably expressed on cell surfaces
6. MHC cannot discriminate bt foreign Ags (peptides derived from foreign/microbial proteins) and self Ags(peptides from individual’s own proteins)
7. Body does not react to self-Ags (autoimmune response)

Question 23

2. Each molecule can present many different peptides – MHC has broad specificity for peptide binding

Answer 23

o As long as pockets of MHC can accommodate the anchor residues of the peptide – can be displayed on MHC molecule
o Only 1 or 2 residues of peptide have to fit into cleft
o Each molecule can bind many but not all possible peptides
o Each individual has only a few diff MHC molecules that can present a vast number and variety of Ags

Question 24

3. MHC molecules only bind peptides

Answer 24

o MHC-restricted CD4+ and CD8+ T cells can recognise and respond to protein Ags (source of peptides)

Question 25

4. MHC acquire peptide cargo during biosynthesis and assembly inside cells

Answer 25

o MHC display peptides derived from microbes inside host cells o Thus MHC-restricted T cells recognise cell-assoc microbes o Class I acquire peptides from cytosolic proteins o Class II acquire peptides from proteins in intracellular vesicles

Question 26

5. Only peptide-loaded MHC molecules are stably expressed on cell surfaces

Answer 26

o “empty” MHC are degraded inside cells o Ensure that only “useful” MHC molecules (those displaying peptides) are expressed on cell surfaces for recognition by T cells o Once peptides bind to MHC and are displayed on cell surface, stay bound for a long time (days) – slow off-rate ensures tt after an MHC acquires a peptide, it’ll display it long enough to maximise chance tt it’s specific T cell will recognise it and initiate response

Question 27

6. MHC cannot discriminate bt foreign Ags (peptides derived from foreign/microbial proteins) and self Ags(peptides from individual’s own proteins)

Answer 27

o MHC can display both foreign and self Ags o Although self Ags is always more than foreign Ags, MHC are not all occupied by self Ags as MHC are constantly being synthesised and ready to accept peptides -> adept at capturing any peptides present in cells o T cells also only need to see a peptide presented by a few MHC molecules to initiate immune response

Question 28

7. Body does not react to self-Ags (autoimmune response)

Answer 28

o T cells specific for self-Ags are killed or inactivated | o MHC molecules presenting self Ags is key to normal surveillance fxn of T cells

Question 29

Antigen processing

Answer 29

 Class I MHC o Proteins in cytoplasm of any nucleated cell processed in cytoplasm o Display endogeneously produced peptides o Usually presents self-Ags o Present viral Ags when host cell is infected by virus and produces viral proteins  Class II MHC o Extracellular proteins internalised by specialised APCs (DCs, macrophages, B cells) processed in vesicles o Displayed engulfed proteins  2 different pathways of Ag processing allows sampling of all the proteins present in extracellular and intracellular envs

Question 30

Processing of internalised Ags for display by Class II MHC

Answer 30

1. Mechanisms of internalisation of extracellular microbes/proteins by APCs o Microbes bind to surface receptors specific for microbial products o Microbes bind to receptors tt recognise Abs o Microbes bind to products of complement activation attached to microbes o B lymphocytes: internalise protein tt specifically binds to cells’ Ag receptors o Pinocytosis / Phagocytosis (w/o specific recognition) 2. After internalisation into APCs o Microbial proteins enter acidic intracellular vesicles – endosome/phagosome o Vesicles may fuse with lysosomes o Proteins are broken down in vesicles by proteolytic enzymes into peptides of varying lengths and sequences ``` 3. Synthesis of class II MHC by APCs o Class II MHC synthesised in ER o Each molecule carries an attached protein – invariant chain – contains sequence Class II Invarian Chain Peptide (CLIP) that binds tightly to peptide binding cleft of MHC o “occupied” MHC molecule is then transported to cell surface in an exocytic vesicle which fuses with endosomal vesicle containing peptides derived from ingested extracellular proteins o Endosomal vesicle contains DM protein – remove CLIP from MHC thus making peptide binding cleft available ``` ``` 4. Fate of class II MHC molecule after CLIP is removed o If MHC can bind peptide generated from ingested proteins -> complex becomes stable -> delivered to cell surface o If MHC does not bind peptide -> empty molecule is unstable -> degraded by proteasomes in endosomes o 1 protein Ag can give rise to many peptides but only a few can bind to MHC o Peptides that can bind to MHC and stimulate immune responses: immunodominant epitopes of the Ag ```

Question 31

Processing of cytosolic Ags for display by Class I MHC

Answer 31

1. Ag proteins produced in cytoplasm from o Viruses living inside infected cells o Phagocytosed microbes that may leak from/transported out of vesicles into cytoplasm o Mutated/altered host genes in tumours 2. Proteins are targeted for destruction by proteolysis o Proteins unfolded o Covalently tagged with multiple copies of peptide ubiquitin o Proteasome action on protein -> cleave and degrade protein into peptides with size and sequence capable of binding class I MHC 3. Binding of peptide (in cytoplasm) to MHC (synthesised in ER) o Transporter associated with Antigen Processing (TAP)  Specialised transport molecule in ER membrane  Binds peptides from cytoplasm  Actively pumps peptides across ER membrane into interior of ER o Newly synthesised class I MHC loosely attached to interior face of TAP o As peptides enter ER, captured by Class I MHC molecules o If binding is of right fit -> complex stabilised and transported to cell surface o Class I MHC-peptide complex  Unavailable to bind other peptides  Can resist proteolysis by endosomal proteases due to stability o If MHC does not bind peptide -> unstable -> degraded 4. Co-evolution of microbe and host o Ways viruses use to block class I MHC pathway of Ag presentation  Remove newly synthesised MHC molecules from ER  Inhibit transcription of MHC genes  Block peptide transport by TAP o By inhibiting class I MHC pathway  Viruses reduce presentation of their own Ags to CD8+ T cells  Evade adaptive immune system o Counterbalanced by NK cells  Recognise and kill virally infected cells which have lost class I MHC exp

Question 32

What is the Significance of MHC-associated Ag presentation?

Answer 32

1. Restriction of T cell recognition to MHC-associated peptides 2. Segregation of class I and II ag presentation pathways ensure the correct, specialised immune response against microbes in diff locations

Question 33

1. Restriction of T cell recognition to MHC-associated peptides

Answer 33

o Ensures that T cells see and respond only to cell-associated Ags (Ags of phagocytosed and intracellular microbes)  MHC are cell membrane proteins  MHC molecules can be loaded with peptides only inside cells, where Ags of phagocytes and intracellular pathogens are present

Question 34

2. Segregation of class I and II Ag presentation pathways ensure the correct, specialised immune response against microbes in diff locations (extracellular/intracellular)

Answer 34

o Extracellular microbes  Captured by APCs including B cells and macrophages  Presented by Class II MHC which are mainly expressed on APCs  CD4 is specific for class II -> class II assoc peptides recognised by CD4+ T cells  CD4+ T cells fxn as helper cells -> help B cells produce Abs, help phagocytes destroy ingested microbes  Eliminate extracellular and ingested microbes but not effective against viruses or other pathogens that replicate in cytoplasm of host cells o Intracellular microbes  Cytosolic Ags processed and displayed by class I MHC  Class I associated peptides recognised by CD8+ T cells which diff into CTLs  CTLs kill infected cells and eradicate infection o Nature of protective immune response to diff microbes optimised by linking features of Ag presentation and T cell recognition  Pathways of processing of vesicular and cytosolic Ags  Cellular exp of Class I and II MHC molecules  Specificity of CD4 and CD8 co-receptors for class II and class I molecules respectively  Functions of CD4+ cells as helper cells; CD8+ cells as CTLs  T cells cannot distinguish bt extracellular and intracellular microbes

Question 35

Ag recognised by B cells and other lymphocytes

Answer 35

 B cells use membrane-bound Abs to recognise Ags  Ags can be o Expressed on microbial surfaces eg. capsular or envelope Ags o In soluble form eg. secreted toxins  In response to Ags, B cells secrete Ab  Abs enter circulation and mucosal fluids -> bind to Ags -> neutralise and eliminate Ags  No need for Ag processing or display  Follicular Dendritic Cells (FDCs) o Cells in B cell-rich lymphoid follicles of lymph nodes and spleen o Display Ags to activated B cells o Ags displayed by FDCs are coated with - Abs - Complement byproducts eg. C3b, C3d o Use Fc receptors to bind Ag-Ab complexes o Use receptors for complement proteins to bind Ags with these proteins attached o B cells that bind Ags with high affinity are selected for  T cells (small number) that recognise non-peptide Ags o Natural Killer T cells (NK-T cells): specific for lipids displayed by class I-like CD1 molecules