Chapter 4 and 5 Ig study questions (Tu): 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)

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 1.18

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

Figure 5.20 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 e.g., IgE

-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

Figure 5.16 We return to effector functions of antibodies in Ch.10 How do you switch from IgM/IgD to other isotypes? Heavy chain constant region defines the isotype

Figure Not to scale and simplified Figure above only shows protein- coding regions. Regulatory elements in DNA provide control of recombination and transcription

Figure L

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. RAG-1 & -2 NOT required Ig Class Switch Recombination (CSR): L

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

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

Ig Class Switch Recombination (CSR) is instructed Unlike V(D)J, which is largely random CSR is initiated by CD40L:CD40 + appropriate cytokine IgG 1 IgG 2a IgG 2b IgG 3 IgAIgE IL-4 ++ IL-5 + IFN  ++ TGF  ++

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

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

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

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

Figure 5.25

“sterile” transcription Ig Class Switch Recombination (CSR):

germline transcript= sterile transcript Ig Class Switch Recombination (CSR):

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

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” Somatic Hypermutation (SHM):

Figure 5.24

Monoclonal antibodies as drugs…

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

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.) I  is the promoter for sterile (noncoding) transcription of C  switch region VDJ Somatic hypermutation (SHM):

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

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

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 Somatic hypermutation (SHM):

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.

Somatic Hypermutation (SHM): The Germinal Center Reaction: 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)

Figure 5.26 HL H – heavy chain locus L –  and light chain loci HL HL H HL H H RAG-1/2 AID

The brick?

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)

Chapter 4 and 5 Ig study questions (Tu): 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?