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Immunology > Molecular Basis of Adaptive Immunity (7-11) > Flashcards

Molecular Basis of Adaptive Immunity (7-11) Flashcards

1
Q

What is the structure of an antibody?

A
  • antigen binding site, two identical light chains and two identical heavy chain
    – each has a v region and a c
    region
  • Fc region, interacts with FcRs and complement
  • hinge region, allows binding of antigen in different orientations
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2
Q

How is it determined if a BcR is secreted or membrane bound?

A

differential splicing
if sequence is cut after secretion coding sequence, the terminus is hydrophilic so is secreted
if sequence is cut after membrane coding sequence, terminus is hydrophobic so interacts with membrane
only happens for heavy chain as light chains do not interact w membrane

differential splicing also determines antibody class (IgM or IgD)

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3
Q

What is the function of IgM?

A

low affinity but high avidity, forms pentamers allowing 10 binding sites
good at fixing complement
can be transported across mucosal mebranes, J chain

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4
Q

What is the function of IgG?

A

has 4 subclasses, 1-4
- IgG1 = opsonisation
- IgG3 = complement activation
synthesised at a v high rate, predominant Ig in blood

much higher concentration in secondary response

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5
Q

What is the function of IgA?

A

present in external secretions
primary function is neutralisation
10% of serum
cross links pathogens in gut to prevent them crossing epithelia
has J chain in a dimer, allows for v high environmental resistance

important in mucosal immunity

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6
Q

What is the function of IgE?

A

sole function is binding to FcεR1 on specialised cells eg mast cells
leads to cellular activation and release of
- histamines
- serotonin
- proteases

mediator of allergy and infection w parasitic worms

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7
Q

What is antibody neutralisation?

A
  • blocking the binding of toxins to cells
  • blocking of binding of pathogen to cell receptor
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8
Q

What is antibody agglutination?

A

crosslinking of antibodies bound to antigen to form clumps of pathogens

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9
Q

What is antibody opsonisation?

A

binding to pathogens makes them easier to identify by phagocytes

function of IgM and IgG (IgG is better)

further enhanced w complement

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10
Q

What is antibody dependent cell cytotoxicity (ADCC)

A

antibodies bind to antigen
Fc receptors on Nk cells (CD16) recognise antibody
NK cell releases cytotoxin granules such as perforin and granzyme
perforin punctures holes in cell membrane allowing granzyme to enter and induce apoptosis

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11
Q

What are examples of experimental antibodies and their functions?

A
  1. rituximab, has several functions in the body. binds to CD20 on cells
    - direct killing
    - complement-mediated cytotoxicity (CDC)
    - ADCC
    - antibody-dependent phagocytosis (ADP)
  2. PD1 blockade, important for controlling T cell responses
    - binding to PD1 receptor on DC causes deactivation of T cell
    - blocking of PD1 by monoclonal antibodies prevents deactivation
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12
Q

What groups of genes make up Ig genes?

A

four groups of genes make up immunoglobulin genes

  • V - variable
  • D - diversity
  • J - joining
  • C - constant

L chain: V, J, and C
composed of either k-chain DNA or gamma chain DNA
– gamma gene has less diversity in mice
– both gamma and k can generate high levels of diversity as they both have multiple V and J genes in humans

H chain: V, D, J, and C

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13
Q

How is the k chain recombined in mice?

A

kappa chain is always recombined first

The kappa locus can generate greater diversity due to the gene arrangement

Each V gene can join to any J gene allowing for a greater number of possible combinations

Accounts for 95% of mouse antibodies due to the increased complexity

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14
Q

How is the lambda chain recombined in mice?

A

the lmbda locus has limited arrangement as it is a much simpler gene that k
each v genes is restricted in what J and C regions it can join onto
accounts for only 5% of antibodies that are formed

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15
Q

How is the heavy chain recombined?

A

happens once for each chromosome once per cell
if failure to create functional protein, cell is marked for apoptosis

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16
Q

How is enzymatic gene rearrangement performed by RAG1/2?

A

every V, D and J gene has conserved flanking sequences

  • recombination signal sequences (RSSs)
  • found at both 5’ and 3’ ends of genes
  1. RAG 1 and 2 recognise conserved heptamer/nonamer sequences of RSSs
  2. RAG1/2 complex at one site (eg D gene) binds to a RAG1/2 complex at another site (eg J gene) forming a loop of DNA
  3. RAG2 cleaves RSS DNA and forms hairpin loops. after RSS but before gene
  4. DNA repair enzymes Ku70/80 proteins bind DNA at ends and stabilise strand break complexes

5a. signal joint is ligated to form a loop, diluted as cell divides
5b. DNA protein kinase and Artemis binds closed loops of DNA covering ends of two sections being recombined. this combination cleaves hairpin loops, creating raggedy ends. TdT adds random nucelotides

signal joints are never replicated and are diluted out during clonal expansion, ensure B cell receptor stays specific to that one type

artemis - cleavage of hairpin loops at random locations
TdT - terminal deoxyribonucleotifdyl transferase

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17
Q

How does VDJ recombination occur before transcription?

A

transcription requires an enhancer which is downstream of genes

transcription cannot start until enhancer is close to promoter so recombination has to occur first

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18
Q

What is affinity maturation and what is the mechanism behind it?

A

IgG, IgA and IgE usually have higher affinity than IgM
due to somatic hypermutations that occur after B cell activation
mediated by induced cytidine deaminase (AID) , a mutator enzyme

AID causes deamination in sequence, C replaced with U which is repaired by RNApol II

this occurs mainly in complimentary defining regions (CDRs) which determine antibody specificity

occurs after clonal expansion and can happen again after reactivation for example during a secondary/tertiary immune response

Kd decreases dramatically after tertiary

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19
Q

What are the mechanisms behind class switching?

A

mature B cells express IgM and IgD as they are first heavy-chain segment in locus
after B cell is activated segement of locus are cut out at switch regions and locus is joined back together, then another segement is first and it is expressed

AID is important for class-switching, by introducing clustered nicks

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20
Q

What is allelic exclusion?

A

only one of two chromosomes is expressed, either maternal or paternal

when rearrangement happens first attempt, same gene on other chromosome is switched off and same attempt is not made again

idk how to explain the rest properly

B cell only has ONE specificity - if it rearranges another specificity on the other chain - could give it self specificity - cause cell with potential pathogen specific BCR to apoptose :(

21
Q

What is the pre-B cell receptor?

A

expressed during the pre-B stage of maturation
composed of μ heavy chain and an invariant surrogate light chain composed of:
1. VpreB protein (homologous to a light chain V domain)
2. lambda 5 protein covalently attached to the μ heavy chain by a disulfide bond

22
Q

What is the Major Histocompatibility Complex?

A

large genomic region that determines transplantation antigens for which there are thousands of genetic variants, alleles, which are matched by tissue typing for transplants to avoid rejection by the immune system

MHC molecules are used by immune system to detect infections inside cells, cancers and threats outside cells

and

high polymorphism is mostly driven by a molecular arms race w pathogens

23
Q

What are classical and non-classical MHC molecules?

A

classical:
- highly polymorphic
- present peptides to T cells
- have wide tissue distribution
- class I expressed on all nucleated cells
- class II expressed on professional and facultative APCs

non- classical
- differ in one or more of classical molecule characteristics
- are derived from classical molecules at different times in evolution

24
Q

Explain the presentation of MHC class I peptides to CD8 T cells.

A

CD8 T cells recognise class I molecules that bind peptides predominantly from the cytoplasm and contiguous structures like the

ABC transporters called TAPs transport peptides into lumen of endoplasmic reticulum which are then loaded onto MHC class I molecules with the help of a chaperone (tapasin), holds MHC class I until right peptide (viral) comes along

then goes to the surface and recognised by CD8-alpha-beta T cells which is in complex with CD3 (a co-receptor) specific to class I

25
Q

Explain the presentation of MHC class II peptides to CD4 T cells.

A

CD4 T cells recognise class II molecules that bind peptides predominantly from intracellular vesicles and the extracellular space
MHC class II system is more complex as more pathogens are recognised compared to MHC class I which just recognises viruses

MHC class II molecules that are involved in presenting processed antigens are synthesised in the endoplasmic reticulum of an antigen presenting cell (APC)
Attached to these molecules is a protein called the class II-associated invariant chain peptide (CLIP), which binds in the peptide-binding site, this folds and stabilises the molecule, which is then translocated to the cell membrane in an exocytic vesicle, where it fuses with an endosomal vesicle that contains processed antigenic peptides
A HLA-DM molecule removes CLIP and enables a peptide to be loaded onto the MHC class II molecule ready to be presented to a CD4+ T cell.

26
Q

How do NK cells protect against viruses in the context of MHC class I molecule expression?

A

NK cells recognise when cells do not have enough MHC class I on their surface which happens in cases of virally infected cells where class I expression has been down-regulated
- scans cells and kills them if they do not have enough

27
Q

What differences are there between MHC class I and II domain structure and gene organisation?

A

MHC class I:
- heavy alpha chain, has transmembrane domain
- beta 2 microglobulin, not bound to cell membrane
- only class I gene is in MHC, beta2m gene is outside MHC

MHC class II
- has alpha chain and beta chain, both have transmembrane domain
- A/B gene pairs are both on MHC

both MHC I and II have same number of disulfide bonds

28
Q

What differences are there between the structure of peptides in which MHC class I and II bind?

A

class I:
- peptides bound to class I have a few anchor residues which bind to deep(er) pockets, B and F
- v specific binding sites
- specific sizes of peptides

class II:
- peptides lie flat and hang out the ends w multiple anchor residues which are less specific
- longer motifs bulge in the middle

29
Q

Are there more classical or non-classical MHC molecules?

A

in mammals, there are just a few classical class I molecules but there can be many more non-classical class I-like molecules

30
Q

Where do T cells arise from and where do they develop?

A

arise from haematopoietic cells in bone marrow
progenitors migrate to thymus where they undergo VDJ recombination

31
Q

What is FoxN1 and how does its absence affect T cell development?

A

TF expressed in thymic epithelial cells important in organisation and development of thymus and T cell development

lack of FoxN1 cause premature involution of thymus and inability to generate T cells

causes nude mice as FoxN1 also controls hair growth

omg captain foxy hiiiiii :3

32
Q

What is the structure of the TCR?

A

contains 2 different chains
- alpha/beta
or
- gamma/delta

held together via disulphide bonds
contains hinge region
short transmembrane region
always membrane bound, never secreted

33
Q

What is a major difference between alpha/beta T cell binding and gamma/delta T cell binding?

A

alpha/beta does not bind antigen alone (MHC restricted)
while gamma/delta is not MHC-restricted

34
Q

What receptors are important for TCR binding?

A

CDR3 is important for binding to peptides (both CD4 and CD8)
CDR1 binds to MHC I
CDR2 binds to MHC II

35
Q

How does alpha/beta TCR undergo VDJ recombination?

A

alpha chain contains V and J
beta chain contains V, D, and J

mechanisms that control recombination events are same between B and T cell
- RAG1/2: cutting at RSS
- Artemis: adding new nucleotides

summary
- RAG1 binds to RSS
- RAG2 is guided by RAG1 and cuts DNA

  • activity of RAG1/2 forms a loop structure and brings part to be joined into close proximity
  • cleavage occurs at ends of coding sequences and loop is cut out
  • artemis cleaves hairpin loops left after cleavage, which creates raggedy ends that are targeted for extension
  • TdT adds new nucleotides to join DNA strands together, which is random so new sequence for every recombination event
36
Q

What are some differences between B and T cell development?

A

T cells do not undergo somatic hypermutation
T cells cannot class switch to different constant regions
T cells do not have a rigid VDJ recombination order
T cells do not increase in specificity after development

both undergo VDJ recombination
both can attempt to rescue a failed recombination event (only on light chain in B cells due to all but D region being removed from genome)

37
Q

How are the TCR gene loci organised?

A

the delta locus is located within the alpha locus, with the V regions interspersed
some of the V segments are shared between both alpha and delta
rearrangement of the alpha chain results in deletion of delta locus from chromosome

RSS order means that D region can be skipped to just join V and J or have two D segments
eg V-J, V-D-J, V-D-D-J

38
Q

How does a T cell become alpha/beta or gamma/delta?

A

recombination of beta, gamma, and delta locus begins simultaneously

first successful rearrangement determines TCR expression
- expression of functional gamma delta receptor suppresses rearrangement of beta receptor
- if beta receptor is successfully rearranged, gamma/delta is suppressed and alpha rearrangement begins

39
Q

How does the pre-TCR develop and mature?

A

pre-T alpha chain joins with the beta chain, forming pre-TCR
allows the beta chain to be expressed on surface ad complex w CD3, which is required for TCR signalling

40
Q

What is the function of the pre-TCR?

A

stimulates the expression of CD4 and CD8 coreceptors
stimulates proliferation
stops additional rearrangements of the beta locus (allelic exclusion)

41
Q

What is positive and negative selection of TCRs?

A

occurs after rearrangement of alpha chain

positive selection: intermediate affinity for self (cells must bind to MHC) = survival
after positive selection, T cell proliferates

negative selection: high affinity for peptide MHC w costimulatory signals = deletion

T cells w high (but not too high) sensitivity will become Treg cells

after selection, T cells lose either CD4 or CD8, depends which MHC it binds to

42
Q

How strong are the interactions between a TCR and an MHC?

A

relatively weak
due to negative selection which helps prevents auto-reactivity in T cells

43
Q

What is the TCR cluster?

A

the TCR (positively charged) forms a cluster w various CD3 dimers, delta-epsilon, gamma-epsilon, zeta-zeta (negatively charged)

each CD3 dimer contains 1-3 ITAM motifs
- tyrosine residues are targets for phosphorylation

during APC interaction - T cells cross-talk, TCRs cljuster at point of contact increasing number fo TCR-MHC interactions
- helps to further increase signalling into T cells driving their activation

44
Q

What is the structure and function of the CD4 and CD8 receptors?

A

CD8 has 2 forms:
- one is a heterodimer, CD8alpha and CD8beta
- the other is a homodimer of the CD8alpah chain

CD4 and CD8 act as accessory molecules to the TCR and can increase TCR sensitivity by approximately 100 fold

45
Q

How do ITAM motifs allow TCR signalling?

A
  • ITAMs have tyrosine residues which are targets for phosphorylation
  • when CD4 or CD8 interact w MHC, p56lck (a kinase) can then interact w the ITAMs to induce activation of T cells
    – CD4s - helper functions
    – CD8s - cytotoxic functions
  • association of p56lck w CD3 helps to further stabilise TCR-MHC interaction
46
Q

What is the immunological synapse?

A

the interaction between TCR and MHC crosstalk

central super molecular activating complex
– TCR/CD3 - MHCI/II
– co-receptors (CD4/CD8)
– co-stimulation (CD28/CD80, CD86)

peripheral super molecular activating complex
– adhesion molecules
- LFA-1/ICAM-1
- CD2/LFA-3

47
Q

Summarise B cell development up to the immature B cell stage.

A
  1. interaction w stromal cells inititates B cell differentiation through IL-7 and E2A
  2. After initiating differentiation to the B cell lineage, the D-J segment of the H cain start rearranging in early pro-B cells

Once D and J have rearranged, early pro-B cells move on to the late pro-B cell stage,and V starts rearranging first on one allele.

  1. after VDJ recombination, first check occurs
    - where cells express surrogate light chain forming the pre-BCR
    - If the VDJ rearrangement in the first allele is successful, the cell moves on. If not, thesecond allele starts rearranging. If this is not successful either, the cell dies. If it issuccessful, the cell moves on to the large pre-B stage
  2. large pre-B cells perfrom allelic exclusion, proliferate, and rearrange the L chain leading to small pre-B stage
    • light chain rearrangement is checked, if successful, allelci exclusion takes place and IgM can be expressed, moving on to immature B cell stage
48
Q
A

Immunology (5 decks)

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