Exam II (generation of antibody diversity + B cell development) Flashcards
opsonin
any molecule that enhances phagocytosis by tagging it for binding to a cell surface
receptor
- complement proteins that bind complement receptors
- antibodies that bind Fc receptors on phagocytic cells
opsonization
“the process by which bacteria are altered by opsonins so as to become more readily and more efficiently engulfed by phagocytes”
what is the difference between an antibody being able to activate complement and acting as an opsonin?
IgG - complement activation and works with the complement receptor to induce phagocytosis
IgG - can also work as an opsonin itself by binding to Fc receptors on surfaces of certain phagocytes
most abundant isotype
IgG
combinatorial diversity
multiple germ line segments
generation of antibody diversity is due to:
- combinatorial diversity
- Junctional diversity
- Somatic hypermutation
human immunoglobin gene germline configuration
- in all cells except for developed lymphocytes, immunoglobin DNA exists in this configuration
- all loci are on 3 different chroosomes
- composed of three types of gene segments V (variable), J (joining) and within the heavy chain locus D, (diversity) segment
two types of light chains
kappa and lambda
a heavy chain will unite with EITHER
kappa OR lambda
Recombination occurs in a precise series of steps
- Heavy chain begins rearrangement. The D to J segments are joined. Next, the joining of the V segment to the DJ segment. This locus is now ready to be transcribed (VDJ)
- After the heavy chain undergoes recombination, the light chain undergoes rearrangement. Since there are no D segments in the light chain, the only recombination is joining the V to J. This locus is now ready to be transcribed. Allelic exclusion also applies to the light chain as well.
antigen binding site heavy chain
VDJ
Antigen binding site light chain
VJ
allelic exclusion
only one B cell receptor will be expressed by any one B cell
applies to both heavy and light chain
Sequence of events during VDJ recombination
- Synapsis
- Cleavage:RAG1/2
- Hairpin Processing: artemis
- Joining
Synapsis
two selected coding segments and their adjacent RSSs are brought together by chromosomal looping
cleavage: RAG1/2
complexes generate ds breaks in DNA, forming hairpin loops
Hairpin processing: Artemis
Artemis opens hairpins at coding ends
Joining
non-homologous end joining
Ku70, Ku80, DNA ligase
RSS
recombination signal sequence
VDJ recombination is reversible/irreversible?
Irreversible
VDJ (heavy chain) and VJ (light chain) can be found in
the majority of the variable region - near amino terminus
types of combinatorial diversity
- multiple gem line gene segments
- multiple heavy and light chain pairings
Junctional diversity occurs at
CDR3 region -> allowing greater variability
junctional diversity increases diversity by
addition of nucleotides.
Can generate antibodies that have different amino acids
TdT
terminal deoxynucleotidyl transferase
TdT funciton
mediates junctional diversity - additional of nucleotides between the junctions of gene segments VDJ
the aa inserted by junctional diversity are inserted
between the V D J junctions (in between these)
N nucleotide addition
are added to both strands, at the junction (by TdT)
can potentially change the reading frame if the nucleotides aren’t a multiple of 3
P nucleotides
nucleotides complementary to the loose/uneven ends
V region assembly from gene fragments – somatic recombination of genomic DNA
irreversible
generation of junctional diversity – imprecision in joining rearranged DNA segments adds non-germline nucleotides (P and N) and deletes germ-line nucleotides
irreversible
assembly of transcriptional controlling elements –promoter and enhancer are brought closer together by V region assembly
irreversible
Transcription activated with coexpression of surface IgM and IgD –two patterns of splicing and processing RNA are used
reversible and regulated
synthesis changes from membrane Ig to secreted antibody –two patterns of splicing and processing RNA are used
reversible and regulated
somatic hypermutation – point mutation of genomic DNA
irreversible
isotype switch – somatic recombination of genomic DNA
irreversible
events that happen in the formation of B cell receptor
- combinatorial diversity
- junctional diversity
event after rearrangement and activation
somatic hypermutation
Phase 1 - generation of diverse and clonally expressed B cell receptors in the bone marrow
repertoire assembly (BM)
Phase 2 - alteration, elimination or inactivation of B cell receptors that binds to components of the human body
negative selection - BM
phase 3 - promotion of a fraction of immature B cells to become mature B cells in the secondary lymphoid tissues
positive selection
( secondary lymphoid organs )
Phase 4 - recirculation of mature B cells between lymph, blood, and secondary lymphoid organs
searching for infection
phase 5 - activation and clonal expansion of B cells by pathogen derived antigens in secondary lymphoid tissues
finding infection
phase 6 - differentiation to antibody secreted plasma cells and memory B cells in secondary lymphoid tissue
attacking infection
sites of B cell development and response
bone marrow - B cell receptor is formed/assembled + NEGATIVE selection
if a B cell is not strongly reactive to self, it will move to secondary lymphoid organs (spleen, lymph node), where they can get activated
sites of B cell development and response
bone marrow - B cell receptor is formed/assembled + NEGATIVE selection
if a B cell is not strongly reactive to self, it will move to secondary lymphoid organs (spleen, lymph node), where they can get activated
stem cell in BM
u germine (H)
K/L germline (L)
Pro B Cell in BM
uDJ (mu - heavy chain)
K/L germline, light chain
Pre B Cell in BM
muVDJ
K/L germline, light chain
pre b cell receptor
heavy chain can be tested for the ability to pair with a light chain - indicates productive rearrangement has occurred
Immature B cell
muVDJ (H)
K/L - VJ
We now have a heavy chain (mu) + light chain fully rearranged
Signal from a properly assembled Pre-B cell receptor induces
allelic exclusion at other heavy chain locus
surrogate light chain
pairs with heavy chain, in order to test the heavy chain (IgB,A next to heavy chain)
Surrogate light chain consists of
VpreB
Lambda 5
invariant molecules
fully formed B cell receptor
has both heavy and light chains + associated with IgBeta and IgAlpha
Immature B cell (in the secondary lymphoid organs)
BEFORE ACTIVATION
alternative splicing to give both delta and mu chains - IgD and IgM
Antigen activated B lymphoblast (in the secondary lymphoid organs)
AFTER ACTIVATION
Alternative splicing to secrete Ig
Isotype switching
Somatic hypermutation
An antigen activated B lymphoblast can become an
antibody secreting plasma cell (lose cell surface expression) OR memory cell (maintain cell surface expression)
somatic hypermutation is induced by
AID
Activation Induced Cytidine Deaminase
somatic hypermutation results in
point mutations, most often within variable regions of immunoglobins after rearrangement
somatic hypermutation leads to
affinity maturation in the antigen-specific B cell pool
Somatic hypermutation gives rise to affinity maturation of the B cell response to antigen
- After activation, B cells undergo clonal expansion
- Some clones will experience mutations in the antigen binding site that enhance antigen binding
- Other clones will sustain mutations that reduce the affinity of the antigen binding site for antigen
selection depends on
T cells! T cells need MHC to be able to recognize antigens.
Increased affinity leads to
increased antigen uptake, processing and presentation.. leading to this cell getting more help from T cells
this cell, with increased affinity, will undergo clonal expansion
transcription produces local single stranded DNA
in B cells, AID attacks cytidine in ssDNA to produce uridine
AID also mediates
isotype switching
isotype switching
changes the constant region of the heavy chain.
c(U), c(d), c(gamma).
last one to be switched to is Calpha2
Isotype or class switching
- happens after B cell activation in proliferating B cells
- like somatic hypermutation, isotype switching is also dependent on AID
- causes irreversible changes in DNA, removing intervening C regions
- However, remaining C regions should still be used in subsequent switches
AID responsible for
somatic hypermutation and isotype switching
S(mu) + Sgamma1
AID has to act at these sites for isotype switching
Lack of RAG leads to
SCID - no T cells or B cells can be produced
Lack of TdT leads to
significantly reduced diversity in B cells repertoire
Lack of AID leads to
no somatic hypermutation or isotype switching, produce only low affinity IgM, called hyper IgM immunodeficiency
Events that rely on changes in DNA
Somatic Recombination
Junctional Diversity
Somatic Hypermutation
Isotype Switching
Events that rely on changes in RNA
Dual expression of IgD and IgM
Expression of transmembrane vs secreted forms of IgM
IgM and IgD (getting rid of either)
are changes in RNA due to differential splicing NOT changes in DNA. All C regions are maintained in the DNA at this point
No class switching has occurred yet.
negative selection
in bone marrow - primary organ
positive selection
secondary organs
