Download T cell independent responses T-independent antigen activate B

Extra office hours:
Thurs, Feb 8 11am-12
Fri, Feb 9 2-4pm
I WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!!
B cell development (antigen dependent)
Dina, Tim, and I encourage all confused students to come to our office
hours and discussion sections so we can try to help un-confuse you.
The GSIs will conduct a review session in our regular class period on Tues
Feb 13, and will hold office hours during class period on Thurs Feb 15.
(Additional GSI office hours also posted on web!)
First midterm: Thurs Feb 15 at 6pm in 155 Dwinelle (not 2050 VLSB as listed
in the original schedule).
Organization of lymphoid organs
T-independent B cell activation
T cell - B cell collaboration
Class switch recombination and somatic hypermutation
Affinity maturation and memory B cells
Midterm will focus on material covered in lectures and will be designed to
be taken in 90 min. (We have the room till 8pm.)
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T cell dependent and independent B cell responses
T cell independent responses
2 signal model: engagement of antigen receptor (BCR,
“signal 1”) is not sufficient to activate B cell. Also need
co-stimulatory signal (“signal 2”).
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Simple, repetitive antigens (often carbohydrates)
Mostly IgM
Modest affinity
No memory
B cells activated by direct BCR crosslinking
B cells can also be activated via Toll-like
receptors (TLRs)
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T-independent antigen activate B
cells by direct BCR aggregation
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Signal transduction by BCR
Ig-α and Ig-β chains become phosphorylated on tyrosine residues,
and then act as docking sites for other proteins, including tyrosine
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kinases. Assembly of large multiprotein complex: “signalosome”
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Signal transduction by BCR can
be modulated by co-receptors.
B cell development (antigen dependent)
Organization of lymphoid organs
T-independent B cell activation
T cell - B cell collaboration
Class switch recombination and somatic hypermutation
Affinity maturation and memory B cells
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T cell - B cell collaboration
T cell dependent B cell response
•Sequence of events:
•Antigen binding to BCR provides “Signal 1” to B cell.
•Antigen is internalized, processed and antigenic
peptides are displayed on MHC for T cell recognition.
•TH (helper T cell) recognizes antigen-MHC complex
via the T cell antigen receptor (TCR): provides “Signal
1” to T cell.
•B7 on B cell binding to CD28 on T cell provides
“Signal 2” to T cell.
•T cell activation leads to up-regulation of CD40L
which bind to CD40 providing “Signal 2” to B cell.
•Cytokine production by activated T cell also help to
activate B cell.
•B cell proliferates and differentiates into antibody
secreting B cell (plasma cell).
•Required for antibody response to complex antigens-- proteins, lipids
•Requires direct, physical B-T interaction
•Involves multiple cell surface receptors on T and B cells
•Both B and T cell must recognize antigen (but not necessarily the
same epitope).
•Both B and T cells need signal 1 (through antigen receptor) and
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signal 2 (co-stimulation)
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Antigen recognition by B cells vs. T cells
B cells (green) interacting with T cells (red) a few hours after
antigen encounter.
Both form their antigen receptors by V(D)J recombination
In intact lymph node at boundary between cortex and paracortex.
~200x real-time.
B cell receptor (BCR) consists of 2 HC and 2 LC (membrane Ig).
T cell receptor (TCR) consists of αβ heterodimer (membrane form
only).
Both signal by associating with signaling complex in membrane:
Ig-α and Ig-β for B cells, CD3 complex for T cells.
B cells can bind intact protein antigen in solution.
T cells bind peptides displayed on the surface of another cell : an
From Okada et al, PLoS Biology 2005 e150 Videos 5 and 6
“antigen presenting cell” (dendritic cell, macrophage, or B cell).
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Antigen encounter drives B cell maturation
B cell development (antigen dependent)
T-independent B cell activation
T cell - B cell collaboration
Class switch recombination
Somatic hypermutation, affinity maturation and
memory B cells
“Naïve” B cell
(IgM, IgD
membrane
Form only)
And review (if we have time)
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Antigen encounter drives B cell maturation
Proliferation
Antibody secretion
Class switch
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Antigen encounter drives B cell maturation
Proliferation
Antibody secretion
Class switch
“Naïve” B cell
(IgM, IgD
membrane
Form only)
“Naïve” B cell
(IgM, IgD
membrane
Form only)
“Activated” B cell
(secreted IgM and other isotypes:
IgA, IgG, IgE)
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V(D)J Rearrangement
VH gene segments
First 96 aa’s of Ig HC
DH gene segments
3-6 aa’s HC
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Relationship between Heavy-chain isotypes and
constant regions in HC DNA
JH gene segments
10-12 aa’s HC CH exons
Antibody Isotypes
Gene
rearrangement
HC locus constant exons
Variable domain
exon
Constant domain
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exons
Note: Isotype names are called: M, D, G,A, E.
Constant regions
exons are called by corresponding greek symbols: µ,
δ, γ, α, ε
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How can a B cell clone produce antibodies
with the identical antigen binding site but
different constant regions?
Alternate mRNA processing:
For secreted vs membrane Ig and for IgM vs IgD.
Class Switch recombination:
For IgG, IgA, IgE
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Membrane associated vs. secreted Ig:
Differential mRNA splicing
Ig heavy-chain gene
promoter
enhancer
S
V
Leader exon
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DJ
J
J
CH1
Variable domain exon
CH2
CH3
M1 M2
CH4
Constant domain exons
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Ig heavy-chain gene
Membrane associated vs. secreted Ig:
Differential mRNA splicing
Switch
promoter
enhancer sequence
S
V
DJ
J
J
CH1
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CH2
CH3
M1 M2
CH4
Leader exon
V
DJ CH1 CH2CH3CH4
Secreted form mRNA
V
DJ CH1 CH2CH3CH4
Membrane-associated form mRNA
Note: mRNA processing, not DNA recombination is occurring. Both secreted and
membrane forms of Ig HC can be made by the same B cell.
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Expression of IgD: Regulated transcriptional
termination & RNA splicing
Other Ig isotypes (IgG, IgA, IgE) are generated by
a second type of somatic DNA recombination
called Class-Switch Recombination (CSR)
AAAAAA
V
DJ
J
J
CH 1 CH 2
Cδ 1 Cδ 2
CH 3 CH 4
µ chain exons
V
Cδ 3 Cδ 4
δ chain exons
DJ CH1CH2 CH3CH4
AAAAAA
V
DJ
J
J
CH 1 CH 2
CH 3 CH 4
V
DJ
Cδ 1 Cδ 2
Cδ 3 Cδ 4
A“Switch site” located 5’ to each CH segment targets the
recombination machinery.
Cδ 1 Cδ 2 Cδ 3 Cδ 4
Note: mRNA processing, not DNA recombination is occurring. Both IgM and
IgD can be made by the same B cell.
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Heavy-chain isotypes-- same variable
domain, different constant domains
Note: DNA recombination is occurring. Once a B cell has switched to make IgG,
it can no longer make IgM. (However, its siblings can.)
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Activities involved in CSR
• DOES NOT require RAG1 or RAG2
• Does require Ku70, Ku80, & DNA-PK
• Requires at least part of each “switch
sequence”
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How can a B cell clone produce antibodies
with the identical antigen binding site but
different constant regions?
Comparison of V(D)J recombination and class
switch recombination (CSR)
Alternate mRNA processing:
secreted vs membrane Ig and for IgM vs IgD.
Same B cell can simultaneously produce sIg, mIg, IgM and IgD.
development in primary lymphoid organs (bone marrow). CSR
• V(D)J recombination occurs as part of antigen-independent
occurs as part of antigen-dependent development in secondary
lymphoid organs (lymph node, spleen).
• V(D)J requires RAG1 or RAG2. CSR does not.
Class Switch recombination:
IgG, IgA, IgE
Progeny of single B cell can produce different isotypes.
• V(D)J recombination is targeted precisely (RSS). CSR occurs
within simple repetitive DNA sequence (switch sequence).
• Both require Ku70, Ku80, & DNA-PK (dsDNA repair pathway).
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Cytokines (interleukins) direct class switching.
B cell development (antigen dependent)
Organization of lymphoid organs
T-independent B cell activation
T cell - B cell collaboration
Class switch recombination
Affinity maturation, somatic hypermutation
and memory B cells
T cells can determine the type of Ig produced by a B cells
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by the type of cytokines they secrete.
Affinity maturation: the increase in the average affinity
of an antisera that occurs during the course of an
immune response or with successive immunizations
1st
immunization 2nd
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Affinity maturation correlates with Somatic Hypermutation
(SHM). Antibodies produced late in an immune response
have point mutations clustered within CDR regions.
3rd
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Average
Affinity
(log scale)
Sequence
alignments of IgG
isolated from B
cells late in an
immune response.
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Time (weeks)
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Somatic
hypermutation
(SHM) increases
progressively during
the course of an
immune response
and correlates with
increased affinity for
antigen
(Note, higher affinity
corresponds to a lower
Kd.)
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Red bars show positions in which nucleotides differ from those found in germline gene DNA segments.
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The distribution of mutations is limited by the V
gene promoter and the intronic enhancer
The mutation rate within this region is 106 times greater
that the normal mutation rate for other genes.
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Link between DNA repair and somatic hypermutation:
Error-prone repair
Why do Ig genes of activated B
cells show such a high rate of
mutation?
dsDNA break
How does this increased mutation
rate lead to increase antibody
affinity?
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Mice with targeted mutation of the AID gene can produce
IgM but not other isotypes (defective in class switching)
Mutation introduced
during DNA repair
Rate= 1 mutation per 1000nt per cell division
(normal mutation rate is 1 per 100,000,000)
Mechanism not fully understood, but requires the enzyme:
Activation-induced cytidine deaminase (AID)
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Mice with targeted mutation of the AID gene are also
defective for somatic hypermutation (Honjo and colleagues 2000)
(Honjo and colleagues 2000)
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Affinity maturation occurs because of somatic hypermutation
and B cell selection in the germinal center
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Mechanism that generates mutations does not discriminate
between mutations that increase or decrease affinity. Yet,
SHM eventually leads to the generation of antibodies with
higher affinity. The key to this paradox is cellular selection.
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Cellular events in germinal
centers:
Somatic mutation: Evolution in “real time”
• Occurs within germinal centers of secondary
lymphoid organs.
• Hypermutation mechanism generates point
mutants in variable domains
• B cells undergoing rapid cell division
• B cells tested for ability to bind to antigen
displayed on follicular dendritic cells
• B cells with best affinity divide more often
• B cells which can’t compete die by apoptosis
Follicular dendritic cells
present antigen to germinal
center B cells in form of
antibody-antigen complexes.
B cells with highest affinity
antibodies compete more
effectively for survival
signals from follicular
dendritic cells.
Note: follicular dendritic cells are NOT related to the dendritic cells (DC) that we
discussed in earlier lectures. FDC are stromal cells, not blood cells, are not major
mediators of innate immunity, and do not present antigens to T cells.
Selected B cells give rise to
high affinity plasma cells and
memory B cells.
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Activated B cells (green) migrating in germinal center. Naïve T and B
cells (red) are shown for comparison.
~200x real-time.
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The phenomenon of
B Cell Memory
Due to presence of “memory B cells” the progeny of B cells that responded to antigen
in primary immunization.
B cell memory can persist for life.
Correlates with higher frequency of specific B cells, and higher affinity of antibodies.
From Allen et al, Science 2007 v315 (5811) p528 Video 2
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Human genetic immunodeficiency illustrate the importance of
memory B cell responses:
Mechanism that generates and maintains B cell memory?
persistence of antigen, longevity of memory B cells?
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Agglutination as a
clinical assay-- Testing
for Rh incompatibility
Disease:
Erythroblastosis
fetalis
X-linked hyper-IgM syndrome:
Patients lack CD40L on T cells.
Defective memory B cell response
lack germinal centers
low affinity antibodies
suseptible to opportunistic pathogens
Cause:
Mother produces IgG that bind to an
antigen (Rh) on RBC of fetus
Detection:
Expose RBC to anti-human Ab and
look for agglutination
Hyper IgM syndrome 2
mutation in AID
failure to undergo somatic hypermutation
increased suseptibility to bacterial infection
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Exposure to fetal blood cells during first pregnancy can induce a memory B cell
response to the Rh antigen.
Genetic Events in
Ig Gene Expression
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•
•
•
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Treatment of mothers
with antibodies to Rh
(RhoGam) at time of
1st delivery can
prevent her from
developing anti-Rh
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antibodies.
* these involve alterations
in the antibody genes
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DNA rearrangements that occur during VDJ
recombination and class switching can activate
proto-oncogenes
B-cell Development and Human Tumors
Antigen Independent
•
V(D)J recombination*
Transcription
Regulated polyadenylation
and RNA splicing
Class switch
recombination*
Somatic hypermutation*
Antigen Dependent
µ+
Pro-B
Pre-B
Leukemia
Ig genes are markers of stage
Transloc/activ of non-Ig genes
Mature B
Plasma Cell
Lymphoma Plasmacytoma
Bcl-2 to Ig rearr.
c-myc to Ig rearr.
Myc-Ig rearr
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A a result of V(D)J recombination every mature B cell expresses a unique antibody.
Encounter with an antigen leads to clonal expansion of B cells with a particular
specificity.
Review
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B Cell Development
Antigen-independent
phase
(bone marrow, fetal
liver)
Antigendependent phase
(spleen, lymph
node)
Molecular
events
V(D)J rearrangement
Class switch,
Somatic
hypermutation
Cellular
events
proB > preB >
mature B cell
development
B cell
activation,
Memory and
plasma B cell
differentiation
B cell development
The preB cell
Ordered gene rearrangements
A model for allelic exclusion
The role of the preBCR in B cell development
B cell tolerance
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Tolerance to self involves both B and T cells and
operates at early and late stages of B cell development
• There are many overlapping mechanisms that ensure self-tolerance.
• Self-reactive B and T cells are eliminated or inactivated during their
development
• Most B cell responses depend on T cell help, so T cell tolerance
helps to ensure that antibodies against self are not generated.
• B cell-intrinsic tolerance mechanisms are especially important for
T-independent B cell responses.
• Somatic hyper-mutation can potentially generate new self-reactive
specificities after B cells encounter antigen.
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How does a B cell know if an antigen is
self or foreign?
• Timing: Antigens that are encountered soon after IgM
expressing B cells first arise in the primary lymphoid organs
tend to induce tolerance.
• Presence of co-stimulatory signal: Antigens that are
encountered in the absence of co-stimulatory signals (signal 1,
but not signal 2) tend to induce tolerance.
• (Note that co-stimulatory signals are directly or indirectly
produced by innate immune responses!)
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B cell tolerance
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Immature vs. mature B cells
• Clonal deletion-- the removal, by apoptosis, of B cells with
self-specific antigen receptors
• Anergy-- the biochemical inactivation of self-specific B cells
• Receptor editing-- ongoing V(D)J recombination resulting in
light-chain replacement and escape from self-reactivity
• Ignorance-- self-specific B cells are present and functional, but
levels are self proteins are insufficient to trigger autoimmunity.
•IgMlo IgDneg
•BCR crosslinking
leads to apoptosis,
not activation
•Subject to “receptor
editing” as a selftolerance mechanism
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Using rearranged Ig transgenic mice to study B
cell tolerance
Receptor Editing: an important
mechanism of B cell self-tolerance
VDJH exons
receptor editing
V!
V!
VJk exons
J!1
J!2
A transgenic model of B cell tolerance
HEL-expressing transgenic
X
HEL=
Hen Egg
Lysozyme
(not exactly
self, but acts as
a self antigen
when expressed
as a transgene.)
Ck exons
J!3
Upstream Vκ to downstream Jκ rearrangement deletes pre-existing
light chain gene.
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Anti-HEL Ig transgenic
CH exons
Rearranged HC and LC chain cloned from a mature B cell and
introduced into the germline via transgenesis to create an Ig
transgenic mouse line. The majority of B cells developing in
these mice express a single, defined Ig.
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B Cell Tolerance:
Evidence for Clonal Anergy
Anergy: B cells expressing self-reactive
Ig are present but are abnormal and nonfunctional.
Double transgenic
Soluble, secreted
Membrane-associated
HEL
HEL
Anergy
(self-reactive B cells are
present but non-functional.)
Clonal Deletion
(Ig expressing B cells are removed)
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B cells from anti-HEL transgenics can secrete anti-HEL Ig when stimulated.
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B cells from double transgenic mice cannot.
Genetic Events in
Ig Gene Expression
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•
•
•
•
•
V(D)J recombination*
Transcription
Regulated polyadenylation
and RNA splicing
Class switch
recombination*
Somatic hypermutation*
* these involve alterations
in the antibody genes
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