L5 - Humoral responses

Question 1

Steps in B cell activation and differentiation

Answer 1

1. B cells develop inbone marrow - precursor rearranges immunoglobulin genes to create BCR
2. cytokines bind and promote survival
3. migration to lymph nodes to wait to be activated

Question 2

what are BCRs

Answer 2

B-cell receptors are cell surafce Immunoglobulins- Ig

same structure as an antibodyb but has a transmembrane region to tether it

Question 3

what part of BCRs are involved in intracellular signalling

Answer 3

BCR alone cannot generate signal

Immunoreceptor tyrosine-based activation motifs (ITAMs)

on Iga and Igb hetrodimer

phosphorylation of tyrosine residues on activation cause intracellular signal

= P13kinase

Question 4

what are antibodies made of (chains and regions)

Answer 4

2 identical L chains + 2 identical heavy chains

each light chain has:
- 1 variable region VL
- 1 consrant region CL

each heavy chain has:
- 1 variable region VH
- 3 constant regions CH1,CH2 and CH3

hinge protein in between

Question 5

what is in the Variable regions of the heavy and light chains that binds to antigens

Answer 5

hypervariable/complimentatiry-determining regions

V region is made of beta-pleated sheets with loops between them

loops bind to antigens

Question 6

how many CDRs regions /hypervariable per chain per antigen binding site (remeber 2 identical binding sites per antibody)

Answer 6

3

so 6 per side of Y

Question 7

what is the orginal ‘germline theory’ for number of antibodies produced and why is this wrong

Answer 7

1 gene per antibody

not enough genes in genome to code for all possible antibodies

= let alone rest of bodies proteins

Question 8

what is the answer to limited genome and unlimited antibodies

Answer 8

VDJ recombination

Question 9

what are the 2 chromosones that code for light chains (heavu chain coded for by a different one)

Answer 9

lambda and kappa

every antibody is 1 kappa/lambda and 1 heavy chain

Question 10

describe VDJ recombination - simple

Answer 10

light chain composed of smashing togther random gene segments :

1 V gene + 1 J gene

heavy chain made of:

1 V gene + 1 J gene + 1 D gene

constant regions join to the new gene and do not partake in recombination = effector function

Question 11

what is the role of Recombination site sequnces (RSS) in VDJ recombination

Answer 11

short DNA sequnces that flank the regions - V.D and J

site of RAG recruitment and where it cuts

Question 12

what is the 12/23 rule in VDJ recombination and how does it ensure thinsg work fine

Answer 12

There are 12 or 23 bases between RSSs

a 12 RSS can only bind to a 23 RSS

heavy chain V + J genes are ONLY 23 spacers

cannot come together

= ensures inclusion of 12 spacer D gene in the middle

Question 13

name of the enzyme complex that undergoes somatic recombination

Answer 13

V(D)J recombinase

Includes:
1. lymphocyte-specific RAG-1 and RAG-2 endonuclease

2. Ku70 and Ku80

Question 14

role of RAG endonucleases

Answer 14

bind to RSS serqunces

recruit andother RSS

cleave between RSS and coding sequnce removing the non-chosen segments

= leaves hairpin loop

Question 15

jucntional diversity mechansim - following on from RSS cleavage by RAG

Answer 15

1. hairpin loops bound by Ku70/80 and recruit artmesis
2. cleaves hairpin loop leaving open ends
3. TDT binds - Terminal deoxynucleotide transferase
4. randomly adds and removes nucleotides to gap until complimentartity found

= junctional diversity at CDR3

5. DNA ligase heals nick

Question 16

why is there increased diversity via junctional diversity at CDR3 compared to the other hypervariable regions

Answer 16

CDR1 and 2 coded by a single segment - a V region

no junctional diversity can occor here due to no cleavage by RAGs

= CDR3 includes V + J (and a D if the heavy chain)
= chance for random nucleotides to be added at end of gene segments

Question 17

summary of VDJ corecomination: combinatorial diversity + jucntional diversity

Answer 17

combinatorial:
multiple V,D and J genes combine
any heavy chain can combine with any light chain to generate antigen-binding site

junctional:
random nucleotides added to ends of genes

Question 18

what can B cells do AFTER encountering an antigen to produce higher affinity antibodies

Answer 18

somatic hypermutation

= T cells once matured never change affinity

Question 19

what is somatic hypermutationn and what enzyme is most involved

Answer 19

B-cells continously undergo 1 or more amino acid changes in the hypervariable regions

AID enzyme - activation induced cytidine deaminase

Question 20

where does somatic hypermutation take place

Answer 20

germinal centres in lymph nodes
1. Light zone:
somatic hypermutation site and rapid division of B-cells

2. Dark zone:
   affinity maturation - cells compete to identify who has highest affinity

Question 21

Describe affinity maturation in steps (thinkl helper T cells)

Answer 21

1. advantageous mutations in B cells bind and present antigen better
2. T follicular helper (Tfh) cells produce selecting cytokines
3. cytokines promote growth and survival

Question 22

B cells can be activated by T-cell dependant or independant pathways - describe the differences

Answer 22

B cells always need 2 signals to be activated

dependant - protein antigens:

1. BCR binds to antigen and presnets it via MHC 2
2. T cells bind via MHC increasing expressiuon of CD40 leukin

= survival signal

independant - non-protein antigen:
1. antigen binding to BCR
2. binding of PAMP to TLR

= produces NFkB transcription factor

Question 23

why do T-cell independant activations of the B cells look like innate responses

Answer 23

No memory cells produced

No affinity maturation or somatic hypermutation

No class switching of antibodies

= short lived plsams cells that cannot clas siwtch or undergo affinity maturation

= T cells provide the signals needd for all these things via cytokines and bidning of CD40

Question 24

term for chaning the Fc region of an antibody

Answer 24

class switching

= no change in affinity (Fab) but changes effector functiom

Question 25

how many types of antibodies are there

name them

Answer 25

5

IgM
IgD
IgG
IgE
IgA

Question 26

put the 5 antibodies in order they are produced in an immune reponse from earliest to latest

Answer 26

IgM / IgD , IgG , IgE , IgA

= M more important than D - D is not fully understood

Question 27

what vstrcuture does IgM form and hown is this useful as an early produced antibody

Answer 27

pentamer via J chain and disulphide bonds

increases avidity = overall strength of interaction with binding due to 10 biunding sites

useful as IgM does not undergo somatic hypermutation due to early produced
- maay not be highest affinity

Question 28

why are both IgM and IgD found on surface of mature B cells and both involved in early response

Answer 28

produced from the same mRNA transcript

alternative splicng decides which is produced

Question 29

which enzyme is involved in class switching AND somatic hypermutation

Answer 29

AID

• Activation Induced cytidine Deaminase

Question 30

how does class switching happen

Answer 30

constant regions of heavy chains have ‘switch regions’ jsut before them

AID cleaves DNA at switch regions = changes antibody isotype

Question 31

role of antibodies

Answer 31

neutralisation
opsonisation

tagging for NK cells - Nk cell binds to the free fc region of antibody

activation of classical complement pathway

allergic response with mast cells - IgE

Question 32

explain the allegric reponse with mast cells and IgE

Answer 32

resting mast cells bind with high affinity to IgE

bind to antigens and cross link

cross link causes release onf granules

= histamines and inflammatory cytokines

Question 33

describe the classical complement pathway caused by IgM antibodies

Answer 33

pentameric IgM binds to bacterial surface

forms ‘staple form’

C1q binds to staple form IgM

= activates pathway eventually producing C3 convertase