Transcript B cell collaboration - Department of Molecular & Cell Biology

Extra office hours:
Thurs, Feb 8 11am-12
Fri, Feb 9 2-4pm
I WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!!
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).
Midterm will focus on material covered in lectures and will be designed to
be taken in 90 min. (We have the room till 8pm.)
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
T cell dependent and independent B cell 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”).
T cell independent responses
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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)
T-independent antigen activate B
cells by direct BCR aggregation
Signal transduction by BCR
Ig-a and Ig-b chains become phosphorylated on tyrosine residues,
and then act as docking sites for other proteins, including tyrosine
kinases. Assembly of large multiprotein complex: “signalosome”
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
T cell - B cell collaboration
•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
signal 2 (co-stimulation)
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).
B cells (green) interacting with T cells (red) a few hours after
antigen encounter.
In intact lymph node at boundary between cortex and paracortex.
~200x real-time.
QuickTime™ and a
Cinepak decompressor
are needed to see this picture.
QuickTime™ and a
Cinepak decompressor
are needed to see this picture.
From Okada et al, PLoS Biology 2005 e150 Videos 5 and 6
Antigen recognition by B cells vs. T cells
Both form their antigen receptors by V(D)J recombination
B cell receptor (BCR) consists of 2 HC and 2 LC (membrane Ig).
T cell receptor (TCR) consists of ab heterodimer (membrane form
only).
Both signal by associating with signaling complex in membrane:
Ig-a and Ig-b 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
“antigen presenting cell” (dendritic cell, macrophage, or B cell).
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
And review (if we have time)
Antigen encounter drives B cell maturation
“Naïve” B cell
(IgM, IgD
membrane
Form only)
Antigen encounter drives B cell maturation
Proliferation
Antibody secretion
Class switch
“Naïve” B cell
(IgM, IgD
membrane
Form only)
Antigen encounter drives B cell maturation
Proliferation
Antibody secretion
Class switch
“Naïve” B cell
(IgM, IgD
membrane
Form only)
“Activated” B cell
(secreted IgM and other isotypes:
IgA, IgG, IgE)
V(D)J Rearrangement
VH gene segments
First 96 aa’s of Ig HC
DH gene segments
3-6 aa’s HC
JH gene segments
10-12 aa’s HC CH exons
Gene
rearrangement
Variable domain
exon
Constant domain
exons
Relationship between Heavy-chain isotypes and
constant regions in HC DNA
Antibody Isotypes
HC locus constant exons
Note: Isotype names are called: M, D, G,A, E.
Constant regions
exons are called by corresponding greek symbols: a
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
Ig heavy-chain gene
promoter
enhancer
S
V
Leader exon
DJ
J
J
Variable domain exon
CH1
CH2
CH3
Constant domain exons
CH4
M1 M2
Membrane associated vs. secreted Ig:
Differential mRNA splicing
Ig heavy-chain gene
Switch
promoter
enhancer sequence
S
V
DJ
J
J
CH1
CH2
CH3
M1 M2
CH4
Leader exon
V
DJ CH1CH2CH3CH4
Secreted form mRNA
V
DJ CH1CH2CH3CH4
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.
Membrane associated vs. secreted Ig:
Differential mRNA splicing
Expression of IgD: Regulated transcriptional
termination & RNA splicing
AAAAAA
V
DJ
J
J
V
CH1 CH2
CH3 CH4
C1
 chain exons
C2
C3
C4
 chain exons
DJ CH1CH2CH3CH4
AAAAAA
V
DJ
J
J
CH1 CH2
V
C1
CH3 CH4
DJ
C2
C3
C4
C1 C2 C3 C4
Note: mRNA processing, not DNA recombination is occurring. Both IgM and
IgD can be made by the same B cell.
Other Ig isotypes (IgG, IgA, IgE) are generated by
a second type of somatic DNA recombination
called Class-Switch Recombination (CSR)
A“Switch site” located 5’ to each CH segment targets the
recombination machinery.
Note: DNA recombination is occurring. Once a B cell has switched to make IgG,
it can no longer make IgM. (However, its siblings can.)
Heavy-chain isotypes-- same variable
domain, different constant domains
Activities involved in CSR
• DOES NOT require RAG1 or RAG2
• Does require Ku70, Ku80, & DNA-PK
• Requires at least part of each “switch
sequence”
How can a B cell clone produce antibodies
with the identical antigen binding site but
different constant regions?
Alternate mRNA processing:
secreted vs membrane Ig and for IgM vs IgD.
Same B cell can simultaneously produce sIg, mIg, IgM and IgD.
Class Switch recombination:
IgG, IgA, IgE
Progeny of single B cell can produce different isotypes.
Comparison of V(D)J recombination and class
switch recombination (CSR)
• V(D)J recombination occurs as part of antigen-independent
development in primary lymphoid organs (bone marrow). CSR
occurs as part of antigen-dependent development in secondary
lymphoid organs (lymph node, spleen).
• V(D)J requires RAG1 or RAG2. CSR does not.
• 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).
Cytokines (interleukins) direct class switching.
T cells can determine the type of Ig produced by a B cells
by the type of cytokines they secrete.
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
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
3rd
10
Average
Affinity
(log scale)
5
Time (weeks)
Affinity maturation correlates with Somatic Hypermutation
(SHM). Antibodies produced late in an immune response
have point mutations clustered within CDR regions.
Sequence
alignments of IgG
isolated from B
cells late in an
immune response.
Red bars show positions in which nucleotides differ from those found in germline gene DNA segments.
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.)
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.
Why do Ig genes of activated B
cells show such a high rate of
mutation?
How does this increased mutation
rate lead to increase antibody
affinity?
Link between DNA repair and somatic hypermutation:
Error-prone repair
dsDNA break
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)
Mice with targeted mutation of the AID gene can produce
IgM but not other isotypes (defective in class switching)
(Honjo and colleagues 2000)
Mice with targeted mutation of the AID gene are also
defective for somatic hypermutation (Honjo and colleagues 2000)
Affinity maturation occurs because of somatic hypermutation
and B cell selection in the germinal center
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.
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
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.
Cellular events in germinal
centers:
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.
Selected B cells give rise to
high affinity plasma cells and
memory B cells.
Activated B cells (green) migrating in germinal center. Naïve T and B
cells (red) are shown for comparison.
~200x real-time.
QuickTime™ and a
YUV420 codec decompressor
are needed to see this picture.
From Allen et al, Science 2007 v315 (5811) p528 Video 2
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.
Mechanism that generates and maintains B cell memory?
persistence of antigen, longevity of memory B cells?
Human genetic immunodeficiency illustrate the importance of
memory B cell responses:
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
Hyper IgM syndrome 2
mutation in AID
failure to undergo somatic hypermutation
increased suseptibility to bacterial infection
Agglutination as a
clinical assay-- Testing
for Rh incompatibility
Disease:
Erythroblastosis
fetalis
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
Exposure to fetal blood cells during first pregnancy can induce a memory B cell
response to the Rh antigen.
Treatment of mothers
with antibodies to Rh
(RhoGam) at time of
1st delivery can
prevent her from
developing anti-Rh
antibodies.
Genetic Events in
Ig Gene Expression
• V(D)J recombination*
• Transcription
• Regulated polyadenylation
and RNA splicing
• Class switch
recombination*
• Somatic hypermutation*
• * these involve alterations
in the antibody genes
B-cell Development and Human Tumors
Antigen Independent
Antigen Dependent

Pro-B
Leukemia
Ig genes are markers of stage
Transloc/activ of non-Ig genes
Pre-B
Mature B
Plasma Cell
Lymphoma Plasmacytoma
Bcl-2 to Ig rearr.
c-myc to Ig rearr.
Myc-Ig rearr
DNA rearrangements that occur during VDJ
recombination and class switching can activate
proto-oncogenes
Review
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.
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
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.
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!)
B cell tolerance
• 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.
Immature vs. mature B cells
•IgMlo IgDneg
•BCR crosslinking
leads to apoptosis,
not activation
•Subject to “receptor
editing” as a selftolerance mechanism
Receptor Editing: an important
mechanism of B cell self-tolerance
receptor editing
Vk
Vk
Jk
Jk
Jk
Upstream Vk to downstream Jk rearrangement deletes pre-existing
light chain gene.
Using rearranged Ig transgenic mice to study B
cell tolerance
VDJH exons
VJk exons
CH exons
Ck 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.
A transgenic model of B cell tolerance
Anti-HEL Ig transgenic
HEL-expressing transgenic
X
HEL=
Hen Egg
Lysozyme
(not exactly
self, but acts as
a self antigen
when expressed
as a transgene.)
Double transgenic
Soluble, secreted
HEL
Anergy
(self-reactive B cells are
present but non-functional.)
Membrane-associated
HEL
Clonal Deletion
(Ig expressing B cells are removed)
B Cell Tolerance:
Evidence for Clonal Anergy
Anergy: B cells expressing self-reactive
Ig are present but are abnormal and nonfunctional.
B cells from anti-HEL transgenics can secrete anti-HEL Ig when stimulated.
B cells from double transgenic mice cannot.
Genetic Events in
Ig Gene Expression
• V(D)J recombination*
• Transcription
• Regulated polyadenylation
and RNA splicing
• Class switch
recombination*
• Somatic hypermutation*
• * these involve alterations
in the antibody genes