extra office hours:thurs, feb 8 11am-12 fri, feb 9 2-4pm i will not be holding office hours on...
Post on 19-Dec-2015
214 views
TRANSCRIPT
Extra office hours: Thurs, Feb 8 11am-12Fri, 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
• 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)
Signal transduction by BCR
Ig- and Ig- chains become phosphorylated on tyrosine residues, and then act as docking sites for other proteins, including tyrosine kinases. Assembly of large multiprotein complex: “signalosome”
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)
•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).
T cell dependent B cell response
QuickTime™ and aCinepak decompressor
are needed to see this picture.
QuickTime™ and aCinepak decompressor
are needed to see this picture.
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.
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 heterodimer (membrane form
only).
Both signal by associating with signaling complex in membrane:
Ig- and Ig- 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
ProliferationAntibody secretion
Class switch
“Naïve” B cell(IgM, IgD membrane Form only)
Antigen encounter drives B cell maturation
“Naïve” B cell(IgM, IgD membrane Form only)
“Activated” B cell(secreted IgM and other isotypes:
IgA, IgG, IgE)
ProliferationAntibody secretion
Class switch
V(D)J Rearrangement
VH gene segmentsFirst 96 aa’s of Ig HC
DH gene segments3-6 aa’s HC
JH gene segments10-12 aa’s HC
CH exons
Generearrangement
Variable domainexon
Constant domainexons
Relationship between Heavy-chain isotypes and constant regions in HC DNA
Note: Isotype names are called: M, D, G,A, E. Constant regions exons are called by corresponding greek symbols:
Antibody Isotypes
HC locus constant exons
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
V DJ J J CH1 CH2 CH3 CH4
Leader exon
M1 M2S
Variable domain exon Constant domain exons
Ig heavy-chain gene
promoter enhancer
V DJ J J
Switch sequence
CH1 CH2 CH3 CH4
Leader exon
M1 M2S
V DJ CH1 CH2 CH3 CH4 V DJ CH1 CH2 CH3 CH4
Secreted form mRNA 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.
Expression of IgD: Regulated transcriptional termination & RNA splicing
V DJ J J CH1 CH2 CH3 CH4 C1 C2 C3 C4
AAAAAA
V DJ J J CH1 CH2 CH3 CH4 C1 C2 C3 C4
AAAAAA
chain exons chain exons
V DJ CH1 CH2 CH3 CH4
V DJ C1 C2 C3 C4
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.)
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
Time (weeks)
AverageAffinity
(log scale)
5
10
1stimmunization 2nd 3rd
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 germ-line gene DNA segments.
(Note, higher affinity corresponds to a lower
Kd.)
Somatic hypermutation
(SHM) increases progressively during
the course of an immune response
and correlates with increased affinity for
antigen
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
Mechanism not fully understood, but requires the enzyme: Activation-induced cytidine deaminase (AID)
Rate= 1 mutation per 1000nt per cell division(normal mutation rate is 1 per 100,000,000)
Mutation introduced during DNA repair
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.
From Allen et al, Science 2007 v315 (5811) p528 Video 2
QuickTime™ and aYUV420 codec decompressor
are needed to see this picture.
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?
The phenomenon of B Cell Memory
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 responselack germinal centerslow affinity antibodiessuseptible to opportunistic pathogens
Hyper IgM syndrome 2mutation in AID failure to undergo somatic hypermutationincreased 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
+Pro-BPre-BMature BPlasma Cell
Antigen IndependentAntigen Dependent
LeukemiaLymphomaPlasmacytomaBcl-2 to Ig rearr.c-myc to Ig rearr.Myc-Ig rearrIg genes are markers of stageTransloc/activ of non-Ig genes
B-cell Development and Human Tumors
DNA rearrangements that occur during VDJ recombination and class switching can activate
proto-oncogenes
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)
Antigen-dependent 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 self-tolerance mechanism
Receptor Editing: an important mechanism of B cell self-tolerance
Upstream V to downstream J rearrangement deletes pre-existinglight chain gene.
V V J1 J2 J3
receptor editing
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.
CH exonsVDJH exons
Using rearranged Ig transgenic mice to study B cell tolerance
Ck exonsVJk exons
A transgenic model of B cell tolerance
Anti-HEL Ig transgenic HEL-expressing transgenic
X
Double transgenic
Membrane-associatedHEL
Soluble, secretedHEL
Anergy(self-reactive B cells are present but non-functional.)
Clonal Deletion(Ig expressing B cells are removed)
HEL=Hen Egg Lysozyme(not exactly self, but acts as a self antigen when expressed as a transgene.)
B Cell Tolerance:Evidence for Clonal Anergy
B cells from anti-HEL transgenics can secrete anti-HEL Ig when stimulated. B cells from double transgenic mice cannot.
Anergy: B cells expressing self-reactive Ig are present but are abnormal and non-
functional.