Chapter 10 Ig study questions: Can you name at least four ways in which CSR and V(D)J recombination differ? What are the substrates (what genes, what areas) for CSR and SHM? What are the key factors? How does AID work? Where does it operate? How are specific class switch events regulated? Why are antibodies used so widely, as lab reagents, diagnostics, therapeutics?

The brick?

The brick? AID

A difference between Ig and TCR: TCR solely functions as an antigen-specific receptor, whereas Ig encodes both antigen receptor and the major effector molecule. Effector functions are mediated by the IgH constant regions.

Consequently, the molecular biology of the BCR/antibody (immunoglobulin) gene loci following activation is much more complex

i.e., there are further recombination events to generate antibodies with specific effector functions (Ig isotype switching), and site-specific mutagenesis that allows the selection of high-affinity antibodies (affinity maturation) Therefore, these processes are unique to B cells…

Immunological phenomena: -Isotype switching -Affinity maturation Happen after stimulation of mature B cells, in the secondary lymphoid organs (LN, spleen, Peyer’s patches). Mostly occur in germinal centers (“the germinal center reaction”)

Figure 1.22

Immunological phenomenon: -Isotype switching Molecular mechanism: -Class switch recombination -Affinity maturation -Somatic hypermutation

e.g., IgE V segment generated by V(D)J recombination is fixed, but can become mutated by SHM (heavy and light) Class switch recombination changes out heavy chain constant regions, replacing IgM/IgD with a “switched” isotype Figure 10.13

-Isotype switching same Ag binding site different heavy chain constant regions and effector properties Ig comes from the same (heavy and light chain) gene loci

Heavy chain constant region defines the isotype How do you switch from IgM/IgD to other isotypes? Figure 5.19

Not to scale and simplified Figure above only shows protein-coding regions. Regulatory elements in DNA provide control of recombination and transcription I showed you this slide a week ago. This should help to visualize what the whole IgH gene locus looks like. This slide shows the organization of the Ig heavy chain locus. Related to Fig.5.5, but more complex

L Note the switch regions. + Figure 10.21

Ig Class Switch Recombination (CSR): When B cells leave the bone marrow, they express IgM and IgD on their surface. During an immune response, B cells may undergo “class switching” - to give rise to B cells producing IgG, IgA or IgE. L RAG-1 & -2 NOT required

Ig Class Switch Recombination (CSR): During class switching, recombination occurs between switch sites. The initial recombination will occur between the m switch site and a downstream switch site. Since there is no switch site associated with the d constant region gene segment, the B cell cannot class switch to IgD. This figure is from a different textbook

Ig Class Switch Recombination (CSR): Class switching does not alter the VDJ sequence and consequently has no effect on antibody specificity. Figure from another textbook Extra-chromosomal DNA is lost/degraded

Ig Class Switch Recombination (CSR) is instructed Unlike V(D)J, which is largely random, CSR is initiated by CD40L:CD40 plus an instructive cytokine signal Figure 10.23

Ig Class Switch Recombination (CSR): IL-4 receptor “sterile” transcription Extra-chromosomal DNA is lost/degraded

Ig Class Switch Recombination (CSR): CSR requires: Activation-induced deoxycytidine deaminase (AID) Uracil DNA glycosylase (UNG)

Properties of AID Expressed mainly in GC B cells Acts on single-stranded DNA AID

Ig Class Switch Recombination (CSR): AID converts C  U at switch sites U removed by UNG, “abasic site” Cut by endonuclease Joining to another switch site

This figure is incorrect to show sterile transcripts coming off of all the switch region promoters, because transcription, and hence switching, is instructed by cytokine signals. Figure 10.21

Figure 10.21

Ig Class Switch Recombination (CSR): “sterile” transcription Figure from another textbook

Ig Class Switch Recombination (CSR): germline transcript= sterile transcript Figure from another textbook

Ig Class Switch Recombination (CSR): Evidence- AID-/- mice & humans UNG knockout mice No CSR

Somatic Hypermutation (SHM): Occurs during antibody response - NOT B cell maturation. During proliferation of B cells in germinal centers, there are increased mutation rates in the sequence encoding the V regions of the BCR (Ab). This will randomly alter (increase or decrease) the affinity of the B cell’s BCR (Ab) for the eliciting antigen. Those B cells with increased affinity for the antigen are selected. As a result, as an immune response proceeds, the affinity of antibody produced increases  “Affinity maturation” Both heavy and light chain genes undego SHM…

Somatic Hypermutation (SHM): Somatic hypermutation is not “directed” Question: HOW are only the “best” B cells selected? Answer: The immune response is ongoing as SHM is happening. Antigen is being cleared and B cells are expanding. There is competition between B cells for Ag binding, processing and presentation to follicular helper T cells in the germinal center (GC-Tfh cells). Meanwhile, cytokines and survival factors are on the decline as the immune response wanes. Only the B cells with the highest affinity survive to become long-lived plasma cells and memory B cells. Figure 10.14

Growing role of monoclonal antibodies as drugs, examples… Scott AM et al., Cancer Immun. 112:14 (2012)

Somatic Hypermutation (SHM): The Germinal Center Reaction: Somatic Hypermutation (SHM): Evidence- AID-/- mice & humans UNG knockout mice Somatic hypermutation uses a similar mechanism No CSR NO SHM

Somatic hypermutation (SHM): Im is the promoter for sterile (noncoding) transcription of Cm switch region VDJ From Martin & Scharff (2002) SHM targets V, not C region;  = 1/1000 bp/division Transcription means RNA polymerase, not DNA polymerase (typo on page 182 of Janeway textbook.)

Properties of AID Expressed only in GC B-cells Acts on single-stranded DNA What is the mechanism of SHM?

Properties of AID Expressed only in GC B-cells Acts on single-stranded DNA DNA replication What is the mechanism of SHM?

Somatic hypermutation (SHM): AID converts C  U at Ig V exon U can be fixed as T by replication, or U removed by UNG, “abasic site” Cut by endonuclease Error-prone repair

Cryosection of lymph node obtained 5 days after secondary antigen injection. Green: ki-67--dividing cells (spatially defines the germinal center). Red: IgM--primary B cell follicle. Yellow: Macrophages. Recap – figure from another textbook

The Germinal Center Reaction: Somatic Hypermutation (SHM): Evidence for a role of AID in SH and CSR: AID-/- mice & humans: no SH or CSR Fibroblasts transfected with AID gene & substrate  SHM & CSR Other B cell-specific functions not required (necessary and sufficient)

L – k and l light chain loci RAG-1/2 H L H L H L H AID AID H L H H H – heavy chain locus L – k and l light chain loci

The brick?

The brick? AID

The brick? AID The window?

The brick? AID The window? Ig loci (light chain V region, heavy V and S)

The brick? AID The window? Ig loci (light chain V region, heavy V and S) Al?

The brick? AID The window? Ig loci (light chain V region, heavy V and S) Al? Natural Selection (APOBECs)

Okay, once you have all these great antibodies of different isotypes, what are they good for? – review of chapter 1 and preview of next Thursday’s lecture.

Overview of humoral immunity Fc receptor/complement binding Figure 4.1 Humoral immunity is mediated by secreted antibodies a. Therefore they can act at a distance from the site of production b. Action in, e.g., blood, mucosal surfaces, gut, lung Antibody functions are triggered by Ag binding to Antibody V regions Most blood protective antibodies made by long-lived plasma cells in BM

Secreted antibodies have multiple effector functions… Figure 10.1

…that you have seen before (in Chapter 1) From chapter 1. Figure 1.28

…that you have seen before (in Chapter 1) From chapter 1. Figure 1.28

Read Chapter 10 for next Thursday! Stuff from today’s lecture will be put in the context of an immune response! Exciting!