Antibody Diversity

Humoral Response • Ag contact Naïve cells primary humoral response • lag pase : clonal selection: differentiation • Ab secreting plasma cells • Memory B cells: G0 phase • Log phase :Antibody peaks: Ig M: Ig G • Secondary response: Memory cells > naïve cells • Original Antigenic sin: vaccination to 1 strain better response to infection with other strain

Humoral Response • • • •

Naive cell : Response 4-7 days :Lag period 7-10 days: peak Varies depending on Ag :Magnitude • Ig M : Isotype • TD,TI :Antigens • Lower :Ab Affinity

• • • •

Memory B cell 1-3 days 3-5days 100-1000 times greater • IgG • TD • Higher

Organization and expression of IG genes • 1010Differentantibodies • Germline theory : germline cells large Ig genes • Somatic theory : few Ig genes :somatic mutation increases no Dryer and Bennet 2 gene 1 polypeptide : revolutionary model Heavy gene :14 constant : c mu /delta/gamma/epsilon/alpha Light lambda :22 lambda 1 and lambda 2 Light kappa 2 no subtypes Experimental evidence: Tonegawa and Hozumi Light chain: V variable J joining C constant Heavy chain : V variable D diversity J joining C constant

Variable Region gene rearrangement • • • • • • •

Ab producing cell : isotype switching :Ag specificity Light chain: any V and J :variable joins to C Heavy chain any V combines with DJ and then to C RNA polymerase binds to leader sequence 5’V Light : kappa or gamma Heavy: Cmu or Cdelta RNA splicing: » introns b/w leader peptide and VDJ » Region b/w VDJ and constant » introns b/w 2 constant regions Cmu and C delta

• Translation • B cell with IgD and IgM

Recombination signal sequence • To ensure proper rearrangment RSS :• Conserved palindromic sequence • Conserved Nonamer AT rich • Intervening sequences :12bp or 23 bp

• 12bp:1 spacer 23bp:2 turn spacer • 1 turn joins only 2 turn • • • •

RSS on 3’of V,3’and 5’ of D and 5of J Kappa:V1spacer+2spacerJ Lambda: V2spacer+1spacer Heavy :V3’ 2spacer+5’ 1spacerD+3’ 1spacer+5’spacerJ

Enzymes in RSS • RSS: recombinase enzymes • RAG1/RAG2: cleaves 1 strand of DNA Formation of hair pin Ds break SS Endonuclease Cutting of Hairpin,addition of Pnucleotides Terminal deoxyribonucleotidyl transferase:TdT Addition of 15nucleotides Ligation of coding sequences Double strand break repair enzymes Productive arrangement/Non Productive rearrangement Allelic exclusion

Generation of Antibody diversity • Multiple germ line VDJ genes 1 individual 51Vh + 27 Dh+ 6Jh 40V kappa+5J kappa 30V lambda+4J lambda Antibody diversity

• Combinational VJ and VDJ joining random rearrangment 51*27*6= 8262 40*5=200 30*4=120

Junctional Flexibility • Joining of signal sequence: precisely and coding sequence : imprecise = Non productive arrangement : apoptosis Productive arrangement : Diversity

Antibody diversity • P addition Recognition of RSS Ss break Hairpin formation Ds break Generaton sites for addition of Pnucleotides

• N addition:15optional • TdT • D..TC ……TA…J…. D..AG……..AT…J…. 2) D…TCGA ……….ATAT…J 3)Optional addition of N

Antibody Diversity • Somatic Hyper mutation – Within somatic cells mutation : new combination

• Association of Heavy and light genes combination of heavy and light genes