Adaptive immunity - T-Cell receptors & MHC proteins

Question 1

What are the 2 types of T-cells /T-cell receptors and how are they defined

Answer 1

• only expressed on membranes, not as soluble proteins
• T helper cells (CD4 +ve)
  
  • Augment immune responses
• T cytotoxic cells (CD8 +ve)
  
  • Specifically kill infected host cells
  • act a bit like NK cells but much more specific
  • only kill host cells infected with a particular pathogen that they can recognise with their specific T-cell receptors
• Receptor structure on both subpopulations is the same!

Question 2

Describe the basic structure of the T cell receptor

Answer 2

• Broadly fab-like structure
• TCR consists of an 𝛼 and 𝛽 chain
• Each chain has a Variable and Constant region
  
  • Extracellular domains of the T cell receptor are homologous to the variable and constant regions of immunoglobulins.
    
    • each V region contains 3 CDRs (hyper variable)
• Stalk segments under constant regions connected by disulfide bonds
• Transmembrane region
• Cytoplasmic tail
  
  • As the TCR is at the membranes of T cells, it has hydrophobic aa residues on c-terminus of the chains
• A subset of T cells (1-5%) express 𝛾 𝜹 (gamma delta) receptors (chains) instead of 𝛼 𝛽
  
  • less diverse
• V𝛼 and V𝛽 domains each have 3 CDRs (1-3)
• CDR3 regions of 𝛼 and 𝛽 chains are the most variable
  
  • same as for antibodies:
    
    • CDR3 loop region is the most variable

Question 3

Can the T- receptor signal by itself?
explain your answer

Answer 3

• Expression of the T-cell receptor on the cell surface requires association with additional proteins.
  
  • when it recognises antigen it has to associate with additional proteins for signalling to occur. it can’t signal by itsself
    
    • these proteins are known collectively as CD3
    • this gives rise to the T-cell receptor Complex containing:
  • The T cell receptor complex:
    
    • 𝛼 and β subunits (TCR)
      
      • which have to associate with →
    • CD3 subunits (ε, δ and γ) and ζ
      
      • E + D, E+Y and 2 Zeta stalks
  • Required for optimal cell surface expression and signalling
  • CD3 subunits contain ITAMs (Immunoreceptor Tyrosine Activation Motifs) in their cytoplasmic regions
    
    • also found in alpha and beta chains that interact with B cell receptors
    • antigen binding → ITAMs phosphorylated → downstream signalling

Question 4

Draw a diagram of the T-cell receptor complex

Answer 4

see lecture notes: 4th tog

Question 5

Describe the TCR genes

Answer 5

• Similar to antibody receptor genes: 2 genes loci for alpha and beta chains
• Chromosome 14:
  
  • contains alpha 2 exons,
  • V region and many J regions and c domains
  • a bit like the light chain of antibodies
• Chromosome 7
  
  • V segments, J segments and D segments and c domains

Question 6

Does somatic V(D)J recombination occur in T cells? if it does, explain its similarities

Answer 6

exact same as B cell gene rearrangement
only difference is that it occurs in the thymus and not the bone marrow

Question 7

Describe the diversity of T cell receptor genes

Answer 7

T cells generally have more gene segments than B cells, creates bigger junctional diversity compared to B cells.

1. Multiple copies of V region gene segment [Vn x Jn/Vn x Dn x Jn]
   V𝛼 ~70 segments, J𝛼 61, V𝛽 = 52, D𝛽 2, J𝛽 13
   whereas for B cell its: max 6, 23, 40
2. 𝛼 x 𝛽 chain combination [Va x Ja] x [Vb x Db x Jb] =~6*10^6
3. Junctional diversity = ~2*10^11
   
   1. Concentrated in the CDR3s of TCR 𝛼 and 𝛽 chains
      
      1. i.e. these are the most variable
      2. CDR1 and 2 are encoded in germline
      3. CDR3 is the VJ/VDJ join so is very diverse
   2. Total diversity = ~10^18

Question 8

Compare the differences in diversity of B and T cell repertoire

Answer 8

B-cells undergo somatic hypermutation which T-cells don’t, however:
The T-cell repertoire is more diverse because T cells generally have more gene segments than B cells -> creates bigger junctional diversity compared to B cells
T-cell repertoire: 10^18
B-cell repertoire: 10^14

Question 9

What is the main big difference between B-cell antibodies and TCRs?

Answer 9

The V regions of TCRs do NOT undergo somatic mutation!!

Question 10

Why do V regions of TCRs not undergo somatic hypermutation

Answer 10

• why?
  
  • maybe its too dangerous
    
    • if T cells somaticly mutate, you might get receptors recognising own tissues
    • B cells the same may occur but B cells need T cells
  • or they may not just need that high affinity generation
  • real answer: we don’t know for sure

Question 11

Do B and T cells recognise the same antigen? what type of pathogens are they important for dealing with

Answer 11

• B and T cells recognise different types of antigens
• B cell immunity is particularly important in defence against extracellular pathogens
  
  • generally true
  • can bind to structures on surface of pathogens or surface of viruses etc.
• B cells recognise free, “native” antigens
  
  • i.e. non-cell associated antigen (unprocessed antigens)

T cells don’t recognise free, “native” antigens

Question 12

How do T cells recognise antigen?

Answer 12

• T cells also important in dealing with intracellular pathogens
• How can T cells recognise intracellular antigens? TCR can’t look inside the cell so how?
  
  • samples of whatever is inside a cell is displayed on the surface so a T cell wit hthe right receptor can recognise wether or not a cell is infected. so how do they do this?
  • Major Histocompatibility proteins (MHC)
    
    • Display bits of whatever is inside the cell on the surface.
    • If infected, T cell will recognise the non-self presentation i.e. antigen
    • So T cells don’t recognise native antigens, only processed antigens!!
    • antigen must be processed (degraded into smaller peptides)
    • antigen binds to MHC and MHC brings it to the cell surface and presents it
• Protein → peptide → MHC → cell surface → T cell recognition
• T cells recognise “cell-associated, processed antigen
• NB! B cells antibodies react to naive unprocessed antigen, whilst T cells and their TCRs recognise “cell-associated” processed antigen!!!
  
  • T cells require antigen to be presented to them

Question 13

Describe the genes and nature of said genes that encode MHC and their importance

Answer 13

• T cells require antigen presentation by cells expressing Major Histocompatibility proteins (MHC)
• Discovered during research on graft rejection. Encoded by the genes of the Major Histocompatibility Complex Chromosome 6 (in humans)
• also known as HLA molecules in humans (human leucocyte antigen). the genes!! not the protein
  
  • **e.g. HLA-A, HLA-B, HLA-C
  • 3 gene loci coding for 3 different MHC proteins!
• very polymorphic
  
  • e.g. > 1400 alleles of HLA-B locus in the human population. Alleles may differ by up to 20 a.a. substitutions
  • so when you match for transplant, you try to match these genes as close together as possible to avoid graft rejection
• major role in antigen presentation and initiation of T cell responses

Question 14

Describe the concept of MHC restriction

Answer 14

• T lymphocytes can only recognise antigen in the context of self-MHC molecules
  
  • this is known as MHC restriction
• Experiments with inbred mouse strains and virally infected cells (had the same MHC proteins on their surfaces)
• Mouse Strain A and mouse strain B immunised with Virus, T cell from the mice were isolated and cultured in vitro with cells infected with the same virus
  
  • if you took T cells from mouse strain A and mixed with cells from mouse A → would kill mouse A infected cells
  • if you took T cells from mouse strain B and mixed with cells from Mouse B → T cells can’t kill infected cells from mouse A
  • SO T cells will only recognise antigen thats being presented to them by ?? (31 min)
• why did this happen? → 2 ideas
  
  • 2 receptors on T cells – one (TCR) for antigen, one for MHC?
    
    • 1 thats recognised antigen, and one recognising MHC
  • 1 receptor on T cells (TCR) – recognises antigen + MHC?
    
    • only 1 receptor on t cell that recognises antigen and MHC

x-ray crystallography proved answer
- Unknown peptide antigen bound as part of the structure
- At the tip of the molecule in a groove, there was a peptide → proved that T cells recognise MHC and foreign peptide.
- MHC, they bind foreign peptide and transport it to the cell surface

Question 15

Describe the structure of a TCR binding MHC1+peptide

Answer 15

• FAB with CDRs in the middle, MHC protein on the bottom. Peptide in yellow
• Crystallographic studies demonstrated:
  
  • **(1) MHC binds peptide
  • (2) TCR recognises complex of peptide + self-MHC
• The loops
  
  • CDR1 and CDR2 of T cell receptor bind self MHC
    
    • CDR1 and CDR2 inherited in germline so less variable
  • CDR3 binds to the peptide
    
    • hypervariable
• Another explanation idea for why somatic mutation doesnt occur in T cells: This may that if T cell receptor genes mutated → you would loose recognition of self MHC if these CDRs mutate
• CDR1 and CDR2 bind self MHC (germline-encoded)
• CDR3 binds peptide (variation introduced by junctional diversity)

Question 16

Describe the 2 classes of MHC and their roles and what antigen presenting cells are

Answer 16

• Two classes of MHC proteins are involved in antigen recognition by T cells
• MHC class I
  
  • Expressed by all nucleated cells
    
    • so basically all cells apart from red blood cells
  • Present peptides derived from endogenous proteins to cytotoxic (CD8) T cells
    
    • endogenous meaning: has to be synthesised by the cell that will display it
• MHC class II
  
  • Expressed by certain leucocytes (dendritic cells, B cells, macrophages).
    
    • more restricted expression pattern
  • Present peptides derived from exogenous proteins to helper (CD4) T cells
    
    • things taken up by a cell (exogenous)
• Antigen presenting cells: Dendritic cells, B cells and macrophages
  
  • so only the ones that present to T helper cells

Question 17

Describe the protein structure of MHC1 and MHC2

Answer 17

• quite similar
• MHC1:
  
  • Polymorphic transmembrane alpha chain, invariant ß-microglobulin
    
    • invariant = just stabilises molecule doesn’t change
    • alpha chain intrinsic, beta microglobulin just stbilises
    • only alpha is transmembrane
• MHC2:
  
  • Polymorphic transmembrane alpha and beta chains
  • both transmembrane
  • domains further from the membrane → more polymorphic and bind to peptide
• Membrane -proximal domains are Ig-like
  
  • the ones close to membrane
• Membrane distal domains bind peptide
• Membrane distal domains contain polymorphisms

Question 18

Describe how MHC 1 and MHC 2 bind peptides

Answer 18

• • MHCI bind peptides 8-10 a.a. long
    
    • N and C-termini of peptides bind to invariant sites at ends of the groove.
      
      • invariant residues present at the ends of the grooves
    • Two or three “anchor residues” on the peptides bind to “specificity pockets” formed by polymorphic residues.
      
      • at the base of the groove
      • fairly closed (restricts length of peptide that can bind)
  • MHCII bind peptides ~ 13-18 a.a. long
    
    • Peptide backbone interacts with conserved residues that line the base of groove.
    • “Anchor residues” on the peptide bind to “specificity pockets” formed by polymorphic residues at the end of the grooves

Question 19

Does MHC 1 bind bigger or smaller peptide? what’s a good analogy of MHC peptide binding

Answer 19

• • MHC1 smaller peptide
  • MHC II bigger peptide
  • Hotdog analogy:
  • MHC1 is the bun, peptide is the sausage
  • A particular MHC molecule with a particular sequence (allele) can bind a wide range of related peptides (structurally related)
    
    • the diversity of the MHC proteins is inhertited, no somatic recombination etc.
• • MHC1 smaller peptide
  • MHC II bigger peptide
  • Hotdog analogy:
  • MHC1 is the bun, peptide is the sausage
  • A particular MHC molecule with a particular sequence (allele) can bind a wide range of related peptides (structurally related)
    
    • the diversity of the MHC proteins is inhertited, no somatic recombination etc.
• looking from above down onto the molecule
• beta sheets, and alpha helical region coming out of the surface formign the sides of the domain

Question 20

Describe the process of antigen presentation by MHC1

Answer 20

• Antigen presentation by MHCI (endogenous antigen)e.g. virus-infected cell to cytotoxic T cell (CD8 +ve)protein made from within the cell (endogenous)
  • Proteosome:
    
    • multi-subunit complex that breaks down misfolded proteins
    • present in all cells
    • i.e. will try to break down viral proteins too
  • another subunit will join the proteosome creating a → immunoproteosome
  • Immunoproteosome:
    
    • formed when another subunit is produced induced by interferon
  • peptides transported to ER by ATP-hydrolysis driven transporter, TAP (transporter associated with antigen presentation).
  • in ER, antigen can bind MHC1
    
    • MHC1 protein will be destined to be sent up to the cell surface regardless of antigen binding or not
  • peptides loaded onto MHCI in ER
  • MHCI-peptide transported to cell surface for recognition by cytotoxic T cell

Question 21

Describe the process of antigen presentation by MHC2

Answer 21

• Antigen presentation by MHCII (exogenous antigen)
  
  • cell isn’t infected! but needs help from a helper T cell to get the immune response going
  • antigen taken up by phagocytosis or endocytosis → in phagolysosome (if phagocyte) enzymes break down protein of pathogen into peptides → peptides associate with MHC II in the endocytic compartments (vacuoles) (in MHC1 association happens in the ER)→ MHC II take peptides to surface where they can be recognised by T helper cell with the right receptor
  e.g. macrophage/dendritic cell/B cell that has taken up antigen and needs “help” from T helper cell (CD4+ve)
  • antigen taken up by phagocytosis or endocytosis
  • acidification in vesicles promotes unfolding and proteolysis
  • peptides associate with MHC II in the endocytic compartment
  • MHCII-peptide transported to cell surface for recognition by helper T cell

Question 22

Describe cross-presentation by MHC 1 and MHC2

Answer 22

• another type of antigen presentation called cross-presentation
  
  • some circumstances wehre a dendritic cell needs to induce a naive cytotoxic T cell to respond to antigen. The D cell is not itsself infected, infection in other tissue maybe etc, none the less they need to induce the naive T cytotoxic cell to respond
  • not completely understood
  • Exogenous peptide can associate with MHC1
  • shown below: necrotic cell (aren’t infected just dying), dendritic cell may take up a necrotic cell and in endosomal or in the cytosol the antigen can associate with MHC1
  • this is important because it allows anitgen to be presented to Cytotoxic T cells even if the cell itsself isn’t infected → this might be important in the responses Cytotoxic T cells make to some tumours
• CROSS-PRESENTATION
  
  • Some dendritic cells present exogenous peptide associated with MHCI to cytotoxic T cells
• Allows antigen presentation to cytotoxic T cells without the dendritic cells themselves being infected
• Important in cytotoxic T cell responses to many tumours

Question 23

What other roles do MHC proteins have

Answer 23

T cell signalling
Thymic selection

Question 24

Describe the role of MHC proteins in T cell signalling

Answer 24

• also play a role in activation of T cells.
• For T cell activation, in addition to the TCR complex (αβ chains +CD3-proteins (to recognise antigen) + 𝜁 chain), co-receptors are also required to
  
  • 1) stabilise the interaction
  • 2) facilitate signalling
• in MHC2 antigen presentation is CD4, in MHC2 antigen presentation the co-receptor is CD8
• CD4 protein (marker of helper T cells) is able to interact with MHC II itsself, with invariant regions of molecule (region that isn’t polymorphic)
• CD8 protein on cytotoxic T cells can interact with MHC I proteins itsself with invariant regions of molecule
• CD4 and CD8 act as co-receptors for the TCR complex.
  
  • Both contain Ig-like domains.
  • immunoglobulin domains interact with eachother
• CD4/CD8 interact with invariant regions on MHC II/I

Question 25

Describe how MHC interacting with co-receptors leads to T cell signalling?

Answer 25

• Engagement of the CD4/CD8 co-receptors with the TCR complex enhances phosphorylation of the ITAMs, promoting T cell signalling
  
  • how does it enhance signalling: CD4 as example
  • CD4 with MHC protein enahnces signalling because Cterminus of CD4 protein has a protein kinase associated with it called lck tyrosine kinase, which can phosphorylate the iTAM motifs present on CD3 complex associated with TCR.
  • Engagement of the CD4/CD8 co-receptors with the TCR complex enhances phosphorylation of the ITAMs, promoting T cell activation
  • CD4/8 associate with lck tyrosine kinase.

Question 26

Describe the role of MHC proteins in thymic selection

Answer 26

• how T cells are selected in the thymus
• T cells generated in bone marrow, acquire antigen receptors when they are in the thymus
• Thymic selection:
  
  • T cell from bone marrow enters thymus → undergoes somatic recombination of TCR genes → expresses TCR:
  • positive selection: TCR is unable to bind MHC protein = apoptosis
  • negative selection: TCR is able to bind MHC BUT also binds self-peptides e.g. Insulin etc. = apoptosis
  • Thymus is a tissue which may not express proteins like e.g. insulin, so how may you ensure you don’t get selection for insulin if insulin isnt present in the thymus
    
    • this causes type 1 diabetes
    • Genes can express non-thymus proteins through AutoImmune regulator (AIRE)
    • deficienies in the AIRE gene are much more prone to autoimmune disease
    • AIRE genes also expressed in secondary lymphoid tissues, to guard against generation to T cells that recognise self tissues.
  • Thymic selection Ensures T cell only bares a receptor that recognises MHC (crurcial for MHC restriction) you don’t get T cells that react to the bodies own cells and molecules

• Rearrangement of T cell receptor genes ($\alpha \beta$ or $\gamma \delta$)
• MHC selection ($\alpha \beta$)
• AIRE (autoimmune regulator)
  
  • allows expression of non-thymus proteins

Question 27

Describe the MHC genes

Answer 27

HLA = human leukocyte antigen → genes that code for MHC proteins

• discovered during studies on graft rejection
• very polymorphic e.g. HLA-B has ~5000 alleles
  
  • expressed on chromosome 6
• co-dominant expression (i.e. alleles inherited from each parent expressed on cells): allows binding of a wide range of peptides
• NB MHC diversity is inherited and small compared to that of B and T cell receptors
• 3 gene loci for MHC1 protein: HLA-A, HLA-B and HLA-C
  
  • each will code for a polymorphic alpha chain
• 3 gene loci for MHC2 genes: HLA-DP, HLA-DQ and HLA-DR
  
  • will each code for a alpha and beta chain
  • very polymorphic
• we inherit these from our parents, a copy frmo mother and a copy from father, (these are usually different ofc.)
  
  • expressed co-dominantly i.e. alleles inherited from each parent expressed on cells which allows MHC proteins being able to bind a wide range of peptides

Question 28

What are peptide-binding regions? why are they important

Answer 28

• MHC genes allelic variation occurs predominantly in the peptide-binding regions
  
  • MHC genes allelic variation occurs predominantly in the peptide-binding regions
    
    • the bit of the molecule that binds to foreign peptides

Question 29

Why are MHC genes so polymorphic? what are the consequences

Answer 29

• As a human population it means that our MHC proteins (because they are very polymorphic) can recognise lots of different foreign peptides → however there is a drawback to this →
  
  • for all of us, our T cell responses are determined to some extent by which MHC proteins we have on the surface of our cells. although each MHC allele can bind a wide range of peptides, these are still somewhat restricted. We don’t see it much in humans but inbred mice which are identical in MHC proteins, we find that some can respond to certain antigens, whilst some don’t respond to antigens. known as responders and non-responders
  • so MHC polymorphism has evolved in response to pathogens
  • some species show more polymorphism than others e.g. leopards have more restricted polymorphisms (not social species so don’t pass on infections as much)
  • So our ancestors survived the black death, flu epidemic (Spanish flu) and HIV which has influenced the MHC proteins we express
  • so we have evolved as a species to be able to respond to new pathogens

• graft rejection
• ensures wide recognition of foreign peptides BUT
• variability of MHC molecules is small compared to that of TCR (all inherited in genome)
• T cell responses determined by an individual’s MHC type
  
  • each MHC allele can bind a restricted range of related peptides
  • Responders and Non-responders
• MHC polymorphism evolved in response to pathogens
  
  • Black death? Flu? HIV?

Question 30

Name some other functions of MHC protein

Answer 30

• graft rejection
• antigen presentation to T cells
• T cell activation
• development of T cell repertoire/tolerance in thymus
• self/non-self recognition (NK cells “detect” alterations MHCI)
  
  • remember this! from innate immunity
  • some viruses downregulate MHC1 to evade cytotoxic T cells, but then they are now vulnerable to be killed by NK cells
• association with certain autoimmune diseases
  
  • certain expression of some MHC proteins
  • having a particular HLA allele, your risk of developing autoimmune disease can be increased dramatically
• HLA and gender-associated risk for autoimmune disease
  
  • risk increases 87 fold with B27 mutation in HLA allele!!
  • some infections may cause cross interaction with self-proteins