T cell receptors and MHC proteins

Question 1

what are the types of T cell receptors?

Answer 1

T helper cells – CD4+ve: Augment immune responses

T cytotoxic cells – CD8+ve: Specifically kill infected host cells

Question 2

how are TCRs expressed?

Answer 2

only on membranes, not as soluble proteins

Question 3

what is the structure of TCRs?

Answer 3

• C-terminus has hydrophobic amino acids to embed into the membrane
• Alpha chain and beta chain linked by flexible disulphide bond hinge
• Broadly Fab-like structure
• Extracellular domains of the T cell receptor are homologous to the variable and constant regions of immunoglobulins.
• Each V region contains 3 CDRs
  o Vα and Vβ domains each have 3 CDRs (1 – 3)
• CDR3 regions of a and b chains are the most variable.

Question 4

what are the two subsets of TCRs?

Answer 4

alpha-beta TCRs - 95%, more diverse

gamma-delta TCRs - 5%, less diverse

Question 5

what does expression of TCRs on the cell surface require?

Answer 5

Expression of TCR on the cell surface requires association with additional proteins, called CD3

Question 6

what is the TCR complex?

Answer 6

alpha and beta subunits, CD3 subunits (epsilon, delta, gamma) and zeta
- this complex is required for optimal cell surface expression and signalling

CD3 subunit associates closely with the alpha-beta chains of TCR

Question 7

what is the CD3 subunit comprised of?

Answer 7

epsilon, delta, gamma subunits and zeta subunit

ITAMs in its cytoplasmic region which can be phosphorylated to lead to downstream signalling

Question 8

what makes up the TCR genes?

Answer 8

2 gene loci, each for alpha and beta chains
- alpha: chromosome 14, made of 2 exons for V region and lots of J regions (like light chain)
- beta: chromosome 7, made of multiple V exons, J exons and D exons (like heavy chain)

these gene recombine in the thymus

Question 9

how do TCR genes recombine?

Answer 9

Somatic recombination of TCR V region genes:
- Same recombination machinery as that used by developing B lymphocytes.
- Occurs in thymus
- J exon recombined to lie next to a V exon and then the C region
-Rag1 and Rag2 proteins facilitate this in the thymus

Question 10

what processes drive TCR diversity?

Answer 10

1. Multiple copies of V region gene segment [Vn x Jn/Vn x Dn x Jn]
2. alpha x beta chain combination [Va x Ja] x [Vb x Db x Jb] = ~ 6 x 10^6
3. Junctional diversity = ~2 x 10^11
   - Concentrated in the CDR3s of TCR alpha and beta chains

Total diversity = ~ 10^18
- Greater than B cell diversity as they have more gene segments than B cells

Question 11

how are the CDRs encoded for in TCRs?

Answer 11

CDR1 and CDR2 encoded in germline, whereas CDR3 is generated via VDJ recombination

Question 12

why do TCRs not undergo somatic hypermutation?

Answer 12

The V regions of TCRs do NOT undergo somatic mutation
- Possibly too dangerous – high likelihood of TCRs that recognise the body’s own tissues
- Doesn’t need to bind to antigen in free solution like antibody, so may not need to bind with such high affinity
- needs to still recognise MHC proteins to function - mutation may disrupt this

Question 13

what are B cells important for? what kind of antigen do B cells recognise?

Answer 13

B cell immunity is particularly important in defence against extracellular pathogens
- B cells recognise free, native antigens on the surface of pathogens

Question 14

what are T cells important for? what kind of antigen do T cells recognise?

Answer 14

T cells are important in defence against intracellular pathogens e.g. viral infections
- T cells recognise cell-associated, processed antigen

Question 15

how do T cells recognise intracellular pathogens?

Answer 15

Major histocompatibility proteins (MHC):
- protein → peptide → MHC → cell surface → T cell recognition
- Protein is degraded into peptide, which is bound to MHC and transported to the cell surface for recognition
- T cells recognise cell-associated, processed antigen
- Samples of antigenic material inside the infected cell are displayed on the surface to be recognised by the T cell

Question 16

what do T cells require to recognise antigen?

Answer 16

T cells require antigen presentation by cells expressing MHCs

Question 17

what are MHC proteins?

Answer 17

• 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 leukocyte antigen).
• e.g. HLA-A, HLA-B, HLA-C – 3 gene loci encoding 3 different MHCs
• very polymorphic
• e.g. >1400 alleles of HLA-B locus. Alleles may differ by up to 20 a.a. substitutions

Question 18

what is the major role of MHC proteins?

Answer 18

• major role in antigen presentation and initiation of T cell responses

Question 19

what is MHC restriction?

Answer 19

T lymphocytes can only recognise antigen in the context of self-MHC molecules

Question 20

how was MHC restriction discovered?

Answer 20

Experiments with inbred mouse strains and virally infected cells
- Had the same MHC proteins on their surface
- Strains A and B were immunised with a virus and their T cells were isolated and cultured with cells infected with the same virus
- If T cells are taken from strain A and mixed them with cells from mouse A, then the T cells kill the infected cells
- If T cells are taken from strain B, the T cells were unable to kill infected cells from mouse A
- Cannot kill cells from a different strain
- T cells will only recognise antigen presented by self-MHC proteins

Question 21

what were the two theories for MHC restriction?

Answer 21

2 receptors on T cells – one (TCR) for antigen, one for MHC?

1 receptor on T cells (TCR) – recognises antigen + MHC?

Question 22

why does MHC restriction occur?

Answer 22

determined by x-ray crystallography:
- Saw unknown peptide antigen bound as part of the structure
- Proved that T cells recognised self-MHC associated with foreign peptide (antigen)

Question 23

what did crystallographic studies on TCR and MHC demonstrate?

Answer 23

1. MHC binds peptide
2. TCR recognises complex of foreign peptide + self-MHC
3. CDR1 and CDR2 bind self-MHC (germline encoded, which is why T cells don’t undergo somatic mutation)
4. CDR3 binds peptide - variation induced by junctional diversity

Question 24

what are the two types of T cells?

Answer 24

T helper cells - CD4+ve

Cytotoxic T cells - CD8+ve

Question 25

which MHC class interacts with CD8+ve cells?

Answer 25

MHC class I interacts with cytotoxic T cells - Expressed by all nucleated cells - Present peptides derived from endogenous proteins to cytotoxic CD8+ve T cells - Endogenous – protein is synthesised by the infected cell

Question 26

which MHC class interacts with CD4+ve cells?

Answer 26

MHC class II interacts with helper T cells - More restrictive expression pattern - Expressed by certain leukocytes: dendritic cells, B cells, macrophages – antigen-presenting cells - Present peptides derived from exogenous proteins to helper (CD4) T cells - Exogenous – protein taken up from outside the cell

Question 27

what is the structure of MHCI protein?

Answer 27

2 chains: alpha chain and beta chain - polymorphic transmembrane alpha chain - invariant b2-microglobulin to provide stability - polymorphisms are clustered in domains furthest from the membrane which bind to antigen

Question 28

what is the structure of MHCII protein?

Answer 28

2 polymorphic transmembrane alpha and beta chains - Domains closest to membrane are immunoglobulin-like, and furthest are most polymorphic

Question 29

what are the functional domains of MHC proteins?

Answer 29

- Membrane-proximal domains are Ig-like - Membrane-distal domains are bind peptide - Membrane distal domains contain polymorphism

Question 30

how do MHCI proteins bind peptide?

Answer 30

MHCI bind peptides 8-10 amino acids long: - Bottom of groove made of beta-pleated sheets, and alpha-helices curl up the sides of the domain - N and C-termini of peptides bind to invariant sites at ends of the groove. - Two or three “anchor residues” on the peptides bind to “specificity pockets” formed by polymorphic residues at the base of the groove - Fairly closed to restrict length of peptide that can bind

Question 31

how do MHCII proteins bind to peptide?

Answer 31

MHCII bind peptides 13-18 amino acids long: - Peptide backbone interacts with conserved residues that line the base groove - “Anchor residues” on the peptide bind to “specificity pockets” formed by polymorphic residues.

Question 32

what is a proteosome?

Answer 32

multi-subunit complex that breaks down misfolded proteins

Question 33

what is an immunoproteosome?

Answer 33

proteosome induced by interferon

Question 34

how does MHCI present antigen to CD8+ve cells?

Answer 34

e.g virus-infected cell presenting to cytotoxic T cell -Cell that is infected with virus will convert the proteosome to an immunoproteasome to process peptide to the right length (8-10 aa) to interact with MHCI - Peptides transported to ER by ATP-hydrolysis driven transporter, TAP (transporter associated with antigen presentation) - Peptides loaded onto MHCI in ER - MHCI-peptide transported to cell surface for recognition by cytotoxic T cell

Question 35

how does MHCII present antigen to CD4+ve cells?

Answer 35

e.g. macrophage/dendritic cell/B cell taken up antigen and needs help from T helper cell (CD4+ve) - antigen taken up by phagocytosis or endocytosis - phagolysosome breaks down the bacteria - acidification in vesicles promotes unfolding & proteolysis - longer peptides (13-18 aa) associate with MHCII in the endocytic compartment - MHCII-peptide transported to cell surface for recognition by helper T cell

Question 36

what is cross-presentation by MHCs?

Answer 36

- Some dendritic cells present exogenous peptide associated with MHCI to cytotoxic T cells - Triggers naïve cytotoxic T cell to be active - Dendritic cell isn’t actually infected - Allows antigen presentation to cytotoxic T cells without the dendritic cells themselves being infected - Important in cytotoxic T cell responses to tumours

Question 37

what is required for T cell activation?

Answer 37

TCR complex (alpha-beta, CD3, zeta-chain), MHC and co-receptor

Question 38

what is the role of co-receptors on the T cell?

Answer 38

to stabilise the interaction between TCR and MHC to facilitate signalling

Question 39

what is the co-receptor of cytotoxic T cells?

Answer 39

CD8

Question 40

what is the co-receptor of T helper cells?

Answer 40

CD4

Question 41

which co-receptor does MHCII + antigen interact with?

Answer 41

CD4 co-receptor

Question 42

which co-receptor does MHCI + antigen interact with?

Answer 42

CD8 co-receptor

Question 43

how do T cell co-receptors function?

Answer 43

CD4/CD8 interact with invariant regions on MHCII/MHCI - not polymorphic CD4 and CD8 act as co-receptors for the TCR complex. Both contain Ig-like domains Engagement of the CD4/CD8 co-receptors with the TCR complex enhances phosphorylation of the ITAMs, promoting T cell activation - CD4/CD8 C-terminus associates with Lck tyrosine kinase which phosphorylates ITAM motifs on the CD3 complex which is associated with the TCR

Question 44

what is thymic selection?

Answer 44

T cell enters thymus and undergoes somatic recombination of TCR genes - Rearrangement of T cell receptor genes (αβ γδ) - TCR must be able to recognise self-MHC but doesn’t react against self - MHC selection (only in αβ chain T cells) (gamma-delta doesn’t require antigen presentation)

Question 45

what is positive MHC selection?

Answer 45

TCR must bind to self-MHC - T cells which fail this undergo apoptosis

Question 46

what is negative MHC selection?

Answer 46

any TCRs which bind to self-peptide undergo apoptosis

Question 46

how are T cells exposed to non-thymus proteins during thymic selection?

Answer 46

AIRE allows expression of non-thymus proteins in the thymus - e.g. insulin, so that the TCR doesn't recognise self-proteins - prevents autoimmune disease

Question 47

how are MHC proteins encoded?

Answer 47

Encodes by the Major Histocompatibility gene Complex (MHC), expressed on chromosome 6 - MHCI – 3 gene loci: HLA-A,B,C encode for polymorphic alpha chain - MHCII: 3 gene loci: HLA-DP,DQ,DR encode for polymorphic alpha and beta chains - very polymorphic e.g. HLA-B has 5000 alleles

Question 48

how are MHC proteins diverse?

Answer 48

Co-dominant expression (i.e. alleles inherited from each parent expressed on cells): increases no. MHC proteins on surface of cells to allow binding of a wide range of peptides - however MHC diversity is inherited and small compared to that of B and T cell receptors

Question 49

which part of the MHC protein facilitates diversity?

Answer 49

Allelic variation occurs predominantly in the peptide-binding groove - Diversity is in region of molecule that recognises antigen peptide

Question 50

what are the consequences of MHC polymorphism?

Answer 50

- 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 - e.g. Inbred mice have MHC proteins which don’t respond to antigens - MHC polymorphism evolved in response to pathogen - Black death? Flu? HIV?

Question 51

what are the functions of MHC proteins?

Answer 51

- 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) - when they detect self-MHC, NK cells are inhibited from killing - association with certain autoimmune diseases - Choice of mate? – attracted to someone with different MHC proteins from self

Question 52

summarise antigen presentation to T cells:

Answer 52

1. CD8 and CD4 act as co-receptors for the TCR, interacting with MHCI and MHCII, respectively, and contribute to T cell activation. 2. MHC proteins play a role in thymic selection 3. MHC proteins are very polymorphic and co-dominantly expressed, allowing binding of a wide range of peptides 4. An individual’s T cells can only recognise antigen presented by self-MHC molecules (MHC restriction).