Extra office hours: Thurs, Feb 8 11am-12 Fri, Feb 9 2-4pm

I WILL NOT BE HOLDING OFFICE HOURS ON TUESDAY Feb 13!! development ( dependent) Dina, Tim, and I encourage all confused students to come to our office Organization of lymphoid organs hours and discussion sections so we can try to help un-confuse you. T-independent B cell activation 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. - B cell collaboration (Additional GSI office hours also posted on web!) Class switch recombination and First midterm: Thurs Feb 15 at 6pm in 155 Dwinelle (not 2050 VLSB as listed Affinity maturation and memory B cells 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.)

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T cell dependent and independent B cell responses T cell independent responses

• Simple, repetitive (often carbohydrates) • Mostly IgM • Modest affinity • No memory • B cells activated by direct BCR crosslinking 2 signal model: engagement of antigen receptor (BCR, • B cells can also be activated via Toll-like “signal 1”) is not sufficient to activate B cell. Also need receptors (TLRs) co-stimulatory signal (“signal 2”). 3 4

Signal transduction by BCR T-independent antigen activate B cells by direct BCR aggregation

Ig-α and Ig-β chains become phosphorylated on tyrosine residues, and then act as docking sites for other proteins, including tyrosine 5 kinases. Assembly of large multiprotein complex: “signalosom6 e”

1 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 •Required for response to complex antigens-- proteins, lipids which bind to CD40 providing “Signal 2” to B cell. •Requires direct, physical B-T interaction • production by activated T cell also help to •Involves multiple cell surface receptors on T and B cells activate B cell. •Both B and T cell must recognize antigen (but not necessarily the •B cell proliferates and differentiates into antibody same epitope). secreting B cell (). •Both B and T cells need signal 1 (through antigen receptor) and signal 2 (co-stimulation) 9 10

B cells (green) interacting with T cells (red) a few hours after Antigen recognition by B cells vs. T cells antigen encounter. In intact lymph node at boundary between cortex and paracortex. Both form their antigen receptors by V(D)J recombination ~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). 11 12

2 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 “Naïve” B cell memory B cells (IgM, IgD membrane Form only) And review (if we have time)

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Antigen encounter drives B cell maturation Antigen encounter drives B cell maturation

Proliferation Proliferation Antibody secretion Antibody secretion Class switch Class switch

“Naïve” B cell “Naïve” B cell (IgM, IgD (IgM, IgD membrane membrane Form only) Form only) “Activated” B cell (secreted IgM and other isotypes: IgA, IgG, IgE)

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Relationship between Heavy-chain isotypes and V(D)J Rearrangement constant regions in HC DNA

VH gene segments DH gene segments JH gene segments First 96 aa’s of Ig HC 3-6 aa’s HC 10-12 aa’s HC CH exons

Antibody Isotypes

Gene rearrangement

HC locus constant exons Variable domain Constant domain Note: names are called: M, D, G,A, E. Constant regions exon exons 17 exons are called by corresponding greek symbols: µ, δ, γ, α,1 8ε

3 How can a B cell clone produce 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: Ig heavy-chain gene Differential mRNA splicing promoter enhancer

S M1 M2

V DJ J J CH1 CH2 CH3 CH4

Leader exon Variable domain exon Constant domain exons

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Ig heavy-chain gene Membrane associated vs. secreted Ig: Switch Differential mRNA splicing promoter enhancer sequence

S M1 M2

V DJ J J CH1 CH2 CH3 CH4

Leader exon

V DJ CH1 CH2CH3CH4 V DJ CH1 CH2CH3CH4 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.

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4 Expression of IgD: Regulated transcriptional Other Ig isotypes (IgG, IgA, IgE) are generated by a second type of somatic DNA recombination termination & RNA splicing called Class-Switch Recombination (CSR)

AAAAAA C 1 C 2 C 3 C 4 C 1 C 2 C 3 C 4 V DJ J J H H H H δ δ δ δ µ chain exons δ chain exons

V DJ CH1CH2 CH3CH4

AAAAAA

C 1 C 2 C 3 C 4 C 1 C 2 C 3 C 4 V DJ J J H H H H δ δ δ δ A“Switch site” located 5’ to each CH segment targets the

V DJ Cδ1 Cδ2 Cδ3 Cδ4 recombination machinery.

Note: mRNA processing, not DNA recombination is occurring. Both IgM and Note: DNA recombination is occurring. Once a B cell has switched to make IgG,

IgD can be made by the same B cell. 25 it can no longer make IgM. (However, its siblings can.) 26

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”

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How can a B cell clone produce antibodies Comparison of V(D)J recombination and class with the identical antigen binding site but switch recombination (CSR) different constant regions? • V(D)J recombination occurs as part of antigen-independent Alternate mRNA processing: development in primary lymphoid organs (bone marrow). CSR secreted vs membrane Ig and for IgM vs IgD. occurs as part of antigen-dependent development in secondary Same B cell can simultaneously produce sIg, mIg, IgM and IgD. lymphoid organs (lymph node, spleen).

Class Switch recombination: • V(D)J requires RAG1 or RAG2. CSR does not. IgG, IgA, IgE • V(D)J recombination is targeted precisely (RSS). CSR occurs Progeny of single B cell can produce different isotypes. within simple repetitive DNA sequence (switch sequence). • Both require Ku70, Ku80, & DNA-PK (dsDNA repair pathway).

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5 (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 by the type of cytokines they secrete. 31 32

Affinity maturation: the increase in the average affinity Affinity maturation correlates with Somatic Hypermutation of an antisera that occurs during the course of an (SHM). Antibodies produced late in an immune response immune response or with successive immunizations have point mutations clustered within CDR regions.

1st immunization 2nd 3rd

10 Average Affinity Sequence alignments of IgG (log scale) isolated from B cells late in an immune response. 5 Red bars show positions in which nucleotides differ from those found in germ- line gene DNA segments. Time (weeks) 33 34

The distribution of mutations is limited by the V gene promoter and the intronic enhancer 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 mutation rate within this region is 106 times greater 35 that the normal mutation rate for other genes. 36

6 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 Mutation introduced rate lead to increase antibody during DNA repair affinity? 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: 37 Activation-induced cytidine deaminase (AID) 38

Mice with targeted mutation of the AID gene can produce Mice with targeted mutation of the AID gene are also IgM but not other isotypes (defective in class switching) defective for somatic hypermutation (Honjo and colleagues 2000) (Honjo and colleagues 2000)

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Affinity maturation occurs because of somatic hypermutation Mechanism that generates mutations does not discriminate and B cell selection in the 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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7 Cellular events in germinal Somatic mutation: Evolution in “real time” centers:

• Occurs within germinal centers of secondary Follicular dendritic cells lymphoid organs. present antigen to germinal • Hypermutation mechanism generates point center B cells in form of mutants in variable domains antibody-antigen complexes. • B cells undergoing rapid cell division B cells with highest affinity • B cells tested for ability to bind to antigen antibodies compete more displayed on follicular dendritic cells effectively for survival • B cells with best affinity divide more often signals from follicular • B cells which can’t compete die by apoptosis dendritic cells. Note: follicular dendritic cells are NOT related to the dendritic cells (DC) that we Selected B cells give rise to discussed in earlier lectures. FDC are stromal cells, not cells, are not major high affinity plasma cells and mediators of innate , and do not present antigens to T cells. memory B cells. 43 44

Activated B cells (green) migrating in germinal center. Naïve T and B The phenomenon of cells (red) are shown for comparison. ~200x real-time. 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? From Allen et al, Science 2007 v315 (5811) p528 Video 2 45 persistence of antigen, longevity of memory B cells? 46

Agglutination as a Human genetic immunodeficiency illustrate the importance of clinical assay-- Testing responses: for Rh incompatibility Disease: X-linked hyper-IgM syndrome: Erythroblastosis Patients lack CD40L on T cells. fetalis Defective memory B cell response lack germinal centers Cause: low affinity antibodies Mother produces IgG that bind to an suseptible to opportunistic pathogens antigen (Rh) on RBC of fetus

Hyper IgM syndrome 2 Detection: mutation in AID Expose RBC to anti-human Ab and failure to undergo somatic hypermutation look for agglutination increased suseptibility to bacterial infection

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8 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

• V(D)J recombination* • Transcription • Regulated polyadenylation and RNA splicing • Class switch recombination* • Somatic hypermutation* Treatment of mothers with antibodies to Rh • * these involve alterations (RhoGam) at time of in the antibody genes 1st delivery can prevent her from developing anti-Rh 49 50 antibodies.

DNA rearrangements that occur during VDJ B-cell Development and Human Tumors recombination and class switching can activate proto-oncogenes Antigen Independent Antigen Dependent

µ+

Pro-B Pre-B Mature B Plasma Cell Lymphoma

Ig genes are markers of stage Bcl-2 to Ig rearr. Myc-Ig rearr Transloc/activ of non-Ig genes c-myc to 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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9 B Cell Development

Antigen-independent Antigen- phase dependent phase B cell development (bone marrow, fetal (spleen, lymph The preB cell liver) node) Molecular V(D)J rearrangement Class switch, Ordered gene rearrangements events Somatic hypermutation A model for allelic exclusion Cellular proB > preB > B cell The role of the preBCR in B cell development events mature B cell activation, development Memory and B cell tolerance plasma B cell differentiation

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Tolerance to self involves both B and T cells and How does a B cell know if an antigen is operates at early and late stages of B cell development self or foreign? • There are many overlapping mechanisms that ensure self-tolerance. • Timing: Antigens that are encountered soon after IgM • Self-reactive B and T cells are eliminated or inactivated during their expressing B cells first arise in the primary lymphoid organs tend to induce tolerance. development • Most B cell responses depend on T cell help, so T cell tolerance • Presence of co-stimulatory signal: Antigens that are helps to ensure that antibodies against self are not generated. encountered in the absence of co-stimulatory signals (signal 1, • B cell-intrinsic tolerance mechanisms are especially important for but not signal 2) tend to induce tolerance. T-independent B cell responses.

• Somatic hyper-mutation can potentially generate new self-reactive • (Note that co-stimulatory signals are directly or indirectly specificities after B cells encounter antigen. produced by innate immune responses!)

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B cell tolerance • -- the removal, by apoptosis, of B cells with Immature vs. mature B cells 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 •IgMlo IgDneg • Ignorance-- self-specific B cells are present and functional, but •BCR crosslinking levels are self proteins are insufficient to trigger . leads to apoptosis, not activation •Subject to “receptor editing” as a self- tolerance mechanism

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10 Receptor Editing: an important Using rearranged Ig transgenic mice to study B cell tolerance mechanism of B cell self-tolerance

VDJH exons CH exons

receptor editing VJk exons Ck exons

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

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 Upstream Vκ to downstream Jκ rearrangement deletes pre-existing these mice express a single, defined Ig. light chain gene. 61 62

A transgenic model of B cell tolerance B Cell Tolerance: Evidence for

Anti-HEL Ig transgenic HEL-expressing transgenic Anergy: B cells expressing self-reactive Ig are present but are abnormal and non- X functional. HEL= Hen Egg Lysozyme (not exactly self, but acts as a self antigen when expressed Double transgenic as a transgene.) Soluble, secreted Membrane-associated HEL HEL Anergy Clonal Deletion (self-reactive B cells are (Ig expressing B cells are removed) B cells from anti-HEL transgenics can secrete anti-HEL Ig when stimulated. present but non-functional.) 63 B cells from double transgenic mice cannot. 64

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

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