t cell - b cell collaboration class switch recombination...

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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 officehours 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 TuesFeb 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 listedin the original schedule).Midterm will focus on material covered in lectures and will be designed tobe taken in 90 min. (We have the room till 8pm.)

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B cell development (antigen dependent)Organization of lymphoid organsT-independent B cell activation

T cell - B cell collaborationClass switch recombination and somatic hypermutation

Affinity maturation and memory B cells

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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”).4

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)

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T-independent antigen activate Bcells 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 tyrosinekinases. Assembly of large multiprotein complex: “signalosome”

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Signal transduction by BCR canbe modulated by co-receptors.

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T cell - B cell collaborationClass switch recombination and somatic hypermutation

Affinity maturation and memory B cells

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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 thesame epitope).•Both B and T cells need signal 1 (through antigen receptor) andsignal 2 (co-stimulation) 10

•Sequence of events:•Antigen binding to BCR provides “Signal 1” to B cell.•Antigen is internalized, processed and antigenicpeptides are displayed on MHC for T cell recognition.•TH (helper T cell) recognizes antigen-MHC complexvia the T cell antigen receptor (TCR): provides “Signal1” 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 CD40Lwhich bind to CD40 providing “Signal 2” to B cell.•Cytokine production by activated T cell also help toactivate B cell.•B cell proliferates and differentiates into antibodysecreting B cell (plasma cell).

T cell dependent B cell response

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B cells (green) interacting with T cells (red) a few hours afterantigen 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 612

Antigen recognition by B cells vs. T cellsBoth form their antigen receptors by V(D)J recombinationB cell receptor (BCR) consists of 2 HC and 2 LC (membrane Ig). T cell receptor (TCR) consists of αβ heterodimer (membrane formonly).

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“antigen presenting cell” (dendritic cell, macrophage, or B cell).

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B cell development (antigen dependent)

T-independent B cell activationT cell - B cell collaboration

Class switch recombinationSomatic hypermutation, affinity maturation and

memory B cells

And review (if we have time)

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Antigen encounter drives B cell maturation

“Naïve” B cell(IgM, IgD membrane Form only)

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ProliferationAntibody secretion

Class switch


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“Activated” B cell(secreted IgM and other isotypes:

IgA, IgG, IgE)

ProliferationAntibody secretion

Class switch

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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 domain

exons 18

Relationship between Heavy-chain isotypes andconstant regions in HC DNA

Note: Isotype names are called: M, D, G,A, E. Constant regionsexons are called by corresponding greek symbols: µ, δ, γ, α, ε

Antibody Isotypes

HC locus constant exons

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How can a B cell clone produce antibodieswith 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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Ig heavy-chain gene

promoter enhancer

V DJ J J CH1 CH2 CH3 CH4

Leader exon

M1 M2S

Variable domain exon Constant domain exons

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Membrane associated vs. secreted Ig:Differential mRNA splicing

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Ig heavy-chain gene

promoter enhancer

V DJ J J

Switch sequence

CH1 CH2 CH3 CH4

Leader exon

M1 M2S

V DJ CH1CH2CH3CH4 V DJ CH1CH2CH3CH4

Secreted form mRNA Membrane-associated form mRNA

Note: mRNA processing, not DNA recombination is occurring. Both secreted andmembrane forms of Ig HC can be made by the same B cell.

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Membrane associated vs. secreted Ig:Differential mRNA splicing

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Expression of IgD: Regulated transcriptionaltermination & RNA splicing

V DJ J J CH1 CH2 CH3 CH4 Cδ1 Cδ2 Cδ3 Cδ4

AAAAAA

V DJ J J CH1 CH2 CH3 CH4 Cδ1 Cδ2 Cδ3 Cδ4AAAAAA

µ chain exons δ chain exons

V DJ CH1CH2CH3CH4

V DJ Cδ1 Cδ2 Cδ3 Cδ4

Note: mRNA processing, not DNA recombination is occurring. Both IgM andIgD can be made by the same B cell. 26

Other Ig isotypes (IgG, IgA, IgE) are generated bya second type of somatic DNA recombination

called Class-Switch Recombination (CSR)

A“Switch site” located 5’ to each CH segment targets therecombination 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.)

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Heavy-chain isotypes-- same variabledomain, different constant domains

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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 “switchsequence”

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How can a B cell clone produce antibodieswith 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.

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Comparison of V(D)J recombination and classswitch recombination (CSR)

• V(D)J recombination occurs as part of antigen-independentdevelopment in primary lymphoid organs (bone marrow). CSRoccurs as part of antigen-dependent development in secondarylymphoid organs (lymph node, spleen).

• V(D)J requires RAG1 or RAG2. CSR does not.

• V(D)J recombination is targeted precisely (RSS). CSR occurswithin simple repetitive DNA sequence (switch sequence).

• Both require Ku70, Ku80, & DNA-PK (dsDNA repair pathway).

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Cytokines (interleukins) direct class switching.

T cells can determine the type of Ig produced by a B cellsby the type of cytokines they secrete. 32


T cell - B cell collaborationClass switch recombination

Affinity maturation, somatic hypermutationand memory B cells

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Affinity maturation: the increase in the average affinityof an antisera that occurs during the course of an

immune response or with successive immunizations

Time (weeks)

AverageAffinity

(log scale)

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1stimmunization 2nd 3rd

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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.

Sequencealignments of IgG

isolated from Bcells late in an

immune response.

Red bars show positions in which nucleotides differ from those found in germ-line gene DNA segments.

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(Note, higher affinitycorresponds to a lower

Kd.)

Somatichypermutation

(SHM) increasesprogressively during

the course of animmune response

and correlates withincreased affinity for

antigen

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The distribution of mutations is limited by the Vgene promoter and the intronic enhancer

The mutation rate within this region is 106 times greaterthat the normal mutation rate for other genes.

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Why do Ig genes of activated Bcells show such a high rate of

mutation?

How does this increased mutationrate lead to increase antibody

affinity?

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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 introducedduring DNA repair

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Mice with targeted mutation of the AID gene can produceIgM but not other isotypes (defective in class switching)

(Honjo and colleagues 2000)

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Mice with targeted mutation of the AID gene are alsodefective for somatic hypermutation (Honjo and colleagues 2000)

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Affinity maturation occurs because of somatic hypermutationand B cell selection in the germinal center

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Mechanism that generates mutations does not discriminatebetween 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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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 wediscussed in earlier lectures. FDC are stromal cells, not blood cells, are not majormediators of innate immunity, and do not present antigens to T cells.

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Cellular events in germinalcenters:

Follicular dendritic cellspresent antigen to germinal

center B cells in form ofantibody-antigen complexes.

B cells with highest affinityantibodies compete moreeffectively for survivalsignals from follicular

dendritic cells.

Selected B cells give rise tohigh affinity plasma cells and

memory B cells.

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Activated B cells (green) migrating in germinal center. Naïve T and Bcells (red) are shown for comparison.~200x real-time.

From Allen et al, Science 2007 v315 (5811) p528 Video 2 46

Due to presence of “memory B cells” the progeny of B cells that responded to antigenin 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 ofB Cell Memory

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Human genetic immunodeficiency illustrate the importance ofmemory 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 AIDfailure to undergo somatic hypermutationincreased suseptibility to bacterial infection

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Agglutination as aclinical assay-- Testingfor Rh incompatibility

Disease:Erythroblastosis

fetalisCause:

Mother produces IgG that bind to anantigen (Rh) on RBC of fetus

Detection:Expose RBC to anti-human Ab and

look for agglutination

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Exposure to fetal blood cells during first pregnancy can induce a memory B cellresponse to the Rh antigen.

Treatment of motherswith antibodies to Rh(RhoGam) at time of

1st delivery canprevent her from

developing anti-Rhantibodies. 50

Genetic Events inIg Gene Expression

• V(D)J recombination*• Transcription• Regulated polyadenylation

and RNA splicing• Class switch

recombination*• Somatic hypermutation*

• * these involve alterationsin the antibody genes

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µ+

Pro-B Pre-B Mature B Plasma Cell

Antigen Independent Antigen Dependent

Leukemia Lymphoma Plasmacytoma

Bcl-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

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DNA rearrangements that occur during VDJrecombination and class switching can activate

proto-oncogenes

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Review

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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 particularspecificity.

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B Cell Development

B cellactivation,Memory andplasma B celldifferentiation

proB > preB >mature B celldevelopment

Cellularevents

Class switch,Somatichypermutation

V(D)J rearrangementMolecularevents

Antigen-dependent phase(spleen, lymphnode)

Antigen-independentphase(bone marrow, fetalliver)

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B cell developmentThe preB cell

Ordered gene rearrangementsA model for allelic exclusion

The role of the preBCR in B cell developmentB cell tolerance

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Tolerance to self involves both B and T cells andoperates 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 isself or foreign?

• Timing: Antigens that are encountered soon after IgMexpressing B cells first arise in the primary lymphoid organstend to induce tolerance.

• Presence of co-stimulatory signal: Antigens that areencountered 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 indirectlyproduced by innate immune responses!)

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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.

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Immature vs. mature B cells

•IgMlo IgDneg

•BCR crosslinkingleads to apoptosis,not activation

•Subject to “receptorediting” as a self-tolerance mechanism

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Receptor Editing: an importantmechanism of B cell self-tolerance

Upstream Vκ to downstream Jκ rearrangement deletes pre-existinglight chain gene.

V! V! J!1 J!2 J!3

receptor editing

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Rearranged HC and LC chain cloned from a mature B cell andintroduced into the germline via transgenesis to create an Ig

transgenic mouse line. The majority of B cells developing inthese mice express a single, defined Ig.

CH exonsVDJH exons

Using rearranged Ig transgenic mice to study Bcell tolerance

Ck exonsVJk exons

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A transgenic model of B cell toleranceAnti-HEL Ig transgenic HEL-expressing transgenic

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Double transgenicMembrane-associated

HELSoluble, secreted

HELAnergy(self-reactive B cells arepresent but non-functional.)

Clonal Deletion(Ig expressing B cells are removed)

HEL=Hen EggLysozyme(not exactlyself, but acts asa self antigenwhen expressedas a transgene.)

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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-reactiveIg are present but are abnormal and non-

functional.

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Genetic Events inIg Gene Expression

• V(D)J recombination*• Transcription• Regulated polyadenylation

and RNA splicing• Class switch

recombination*• Somatic hypermutation*

• * these involve alterationsin the antibody genes

t cell - b cell collaboration class switch recombination...

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