L5 - Humoral responses Flashcards
Steps in B cell activation and differentiation
- B cells develop inbone marrow - precursor rearranges immunoglobulin genes to create BCR
- cytokines bind and promote survival
- migration to lymph nodes to wait to be activated
what are BCRs
B-cell receptors are cell surafce Immunoglobulins- Ig
same structure as an antibodyb but has a transmembrane region to tether it
what part of BCRs are involved in intracellular signalling
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
what are antibodies made of (chains and regions)
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
what is in the Variable regions of the heavy and light chains that binds to antigens
hypervariable/complimentatiry-determining regions
V region is made of beta-pleated sheets with loops between them
loops bind to antigens
how many CDRs regions /hypervariable per chain per antigen binding site (remeber 2 identical binding sites per antibody)
3
so 6 per side of Y
what is the orginal ‘germline theory’ for number of antibodies produced and why is this wrong
1 gene per antibody
not enough genes in genome to code for all possible antibodies
= let alone rest of bodies proteins
what is the answer to limited genome and unlimited antibodies
VDJ recombination
what are the 2 chromosones that code for light chains (heavu chain coded for by a different one)
lambda and kappa
every antibody is 1 kappa/lambda and 1 heavy chain
describe VDJ recombination - simple
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
what is the role of Recombination site sequnces (RSS) in VDJ recombination
short DNA sequnces that flank the regions - V.D and J
site of RAG recruitment and where it cuts
what is the 12/23 rule in VDJ recombination and how does it ensure thinsg work fine
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
name of the enzyme complex that undergoes somatic recombination
V(D)J recombinase
Includes:
1. lymphocyte-specific RAG-1 and RAG-2 endonuclease
- Ku70 and Ku80
role of RAG endonucleases
bind to RSS serqunces
recruit andother RSS
cleave between RSS and coding sequnce removing the non-chosen segments
= leaves hairpin loop
jucntional diversity mechansim - following on from RSS cleavage by RAG
- hairpin loops bound by Ku70/80 and recruit artmesis
- cleaves hairpin loop leaving open ends
- TDT binds - Terminal deoxynucleotide transferase
- randomly adds and removes nucleotides to gap until complimentartity found
= junctional diversity at CDR3
- DNA ligase heals nick
why is there increased diversity via junctional diversity at CDR3 compared to the other hypervariable regions
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
summary of VDJ corecomination: combinatorial diversity + jucntional diversity
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
what can B cells do AFTER encountering an antigen to produce higher affinity antibodies
somatic hypermutation
= T cells once matured never change affinity
what is somatic hypermutationn and what enzyme is most involved
B-cells continously undergo 1 or more amino acid changes in the hypervariable regions
AID enzyme - activation induced cytidine deaminase
where does somatic hypermutation take place
germinal centres in lymph nodes
1. Light zone:
somatic hypermutation site and rapid division of B-cells
- Dark zone:
affinity maturation - cells compete to identify who has highest affinity
Describe affinity maturation in steps (thinkl helper T cells)
- advantageous mutations in B cells bind and present antigen better
- T follicular helper (Tfh) cells produce selecting cytokines
- cytokines promote growth and survival
B cells can be activated by T-cell dependant or independant pathways - describe the differences
B cells always need 2 signals to be activated
dependant - protein antigens:
- BCR binds to antigen and presnets it via MHC 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
why do T-cell independant activations of the B cells look like innate responses
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
term for chaning the Fc region of an antibody
class switching
= no change in affinity (Fab) but changes effector functiom
how many types of antibodies are there
name them
5
IgM
IgD
IgG
IgE
IgA
put the 5 antibodies in order they are produced in an immune reponse from earliest to latest
IgM / IgD , IgG , IgE , IgA
= M more important than D - D is not fully understood
what vstrcuture does IgM form and hown is this useful as an early produced antibody
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
why are both IgM and IgD found on surface of mature B cells and both involved in early response
produced from the same mRNA transcript
alternative splicng decides which is produced
which enzyme is involved in class switching AND somatic hypermutation
AID
- Activation Induced cytidine Deaminase
how does class switching happen
constant regions of heavy chains have ‘switch regions’ jsut before them
AID cleaves DNA at switch regions = changes antibody isotype
role of antibodies
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
explain the allegric reponse with mast cells and IgE
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
describe the classical complement pathway caused by IgM antibodies
pentameric IgM binds to bacterial surface
forms ‘staple form’
C1q binds to staple form IgM
= activates pathway eventually producing C3 convertase
