ANTIBODY DIVERSITY

STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES COMPLEMENT ACTIVATION BINDING TO CELLS DEGRADATION TRANSPORT Light chain (L) Heavy chain (H) VL CL VH CH Antigen binding Variable domains Antigen Constans domains Effector functions

Multiple myeloma (MM) Plasma cell tumors – tumor cells reside in the bone marrow Produce immunoglobulins of monoclonal origin, serum concentration mg/ml Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification AMINO ACID SEQUENCE OF IMMUNOGLOBULINS 50 kDa Heavy chain 25 kDa Light chain Gel electrophoresis V ariable C onstant Reduction L H

GENETIC BACKGROUND OF ANTIBODY DIVERSITY VL VH Mechanism of the generation of variability? Different rules for encoding the variable and constant regions? S – S

Many GENES ( – ) V2V2V2V2 C2C2C2C2 VnVnVnVn CnCnCnCn V1V1V1V1 C1C1C1C1 1 GEN High rate of somatic mutations in the V-region VC GenGenGenGen Protein 1 GEN = 1 PROTEIN DOGMA OF MOLECULAR BIOLOGY CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE THEORIES

MOLECULAR GENETICS OF IMMUNOGLOUBLINS A single C region gene encoded in the GERMLINE and separate from the V region genes Multiple choices of V region genes available A mechanism to rearrange V and C genes in the genome so that they can fuse to form a complete Immunoglobulin gene. In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be: How can the bifunctional nature of antibodies be explained genetically? This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an individual

The Dreyer - Bennett hypothesis V V V V V V V V V V V V V A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete Immunoglobulin gene C V C A single C region gene is encoded in the germline and separated from the multiple V region genes Find a way to show the existence of multiple V genes and rearrangement to the C gene

Approach Tools: A set of cDNA probes to specifically distinguish V regions from C regions DNA restriction enzymes to fragment DNA Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a plasmacytoma/myeloma) C V V V V V V V V V Germline DNA C V V V V V Rearranged DNA

** * * * B-cell VC V C Embryonal cell V-CmRNA probe CmRNA probe * * The experiment of Susumi Tonegawa 1976

The key experiment of Nobumichi Hozumi and Susumu Tonegawa

There are many variable genes but only one constant gene VCVVV GERM LINE V and C genes get close to each other in B-cells only C V VV B-CELL CONCLUSION PROTEIN GENE REARRANGEMENT OF GENE SEGMENTS INTO A SINGLE FUNCTIONAL UNIT (GENE)

Ig gene sequencing complicated the model The structures of germline V L genes were similar for V , and V, However there was an anomaly between germline and rearranged DNA: Where do the extra 13 amino acids come from? CLCL VLVL ~ 95  ~ 100  L CLCL VLVL ~ 95  ~ 100  JLJL Some of the extra amino acids are provided by one of a small set of J or JOINING regions L CLCL VLVL ~ 208  L

During B-lymphocyte development JkJκJκJκJκJκJκVκVκVκVκVκVκ B-cell 1 JκJκVκVκ B-cell 2 40 Vκ 5 Jκ5 Jκ5 Jκ5 Jκ VκVκVκVκVκVκVκVκ JκJκ JκJκ JκJκ JκJκ Germ line SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS DNA

pA CCκCCκ E JJ Vκ-JκVκ-Jκ VκVκVκVκ P CCκCCκ JVκVκProtein mRNA CCκCCκ JVκVκ AAAA Translation EXPRESSION OF THE KAPPA CHAIN Primary RNA transcript CCκCCκ E JJVκVκ Leader Efficiency of somatic gene rearrangement?

Further diversity in the Ig heavy chain VLVL JLJL CLCL L CHCH VHVH JHJH DHDH L The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes D (DIVERSITY) region Each light chain requires 1 recombination events V L to J L Each heavy chain requires 2 recombination events J H to D H, V H to J H D H,

During B-cell development VH2 JH 65 VH 6 JH VH1VH3 DJH 27 D DDD JH DD SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS DD VH1VH2VH3 VH1VH2

HOW MANY IMMUNOGLOBULIN GENE SEGMENTS Variable (V) Diversity (D)0027 Joining (J)546 Gene segmentsLight chainHeavy chain kappalambda Chromosome 2 kappa light chain gene segments Chromosome 22 lambda light chain gene segments Chromosome 14 heavy chain gene segments IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS

VH D JH VLJL V-Domains C-Domains VH-D-JH VL-JL VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES B cells of one individual

Estimates of combinatorial diversity Taking account of functional V D and J genes: 65 VH x 27 DH x 6JH = 10,530 combinations 40 V  x 5 J  = 200combinations 30 V  x 4 J = 120 combinations = 320 different light chains If H and L chains pair randomly as H 2 L 2 i.e. 10,530x 320 = 3,369,600 possibilities Due only to COMBINATORIAL diversity In practice, some H + L combinations do not occur as they are unstable Certain V and J genes are also used more frequently than others. There are other mechanisms that add diversity at the junctions between genes - JUNCTIONAL diversity GENERATES A POTENTIAL B-CELL REPERTOIRE

Somatic recombination to generate antibody diversity

Severe combined immunodeficiency (SCID) Early manifestation red rash on the face and shoulders, infections with opportunistic pathogens. (Candida albicans, Pneumocystis carnii pneumonia) Lack of palpable lymph nodes Omenn syndrome - RAG deficiency Lack of T-cells and B cells

How does somatic gene rearrangement (recombination) work? 1.How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2.How do V region find J regions and why don’t they join to C regions? rule -Special - Recobnitation Signal Sequences (RSS) - Recognized by Recombination Activation Gene coded proteins (RAGs) PALINDROMIC SEQUENCES HEPTAMERCACAGTGCACAGTGGTGACAC NONAMERACAAAAACCGGTTTTTGT TGTTTTTGGCCAAAAACA

V, D, J flanking sequences V Sequencing upstream and downstream of V, D and J elements revealed conserved sequences of 7, 23, 9 and 12 nucleotides in an arrangement that depended upon the locus VV JJ J D VHVH JHJH 7 9

Recombination signal sequences (RSS) RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS VHVH D JHJH HEPTAMER - Always contiguous with coding sequence NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer VHVH D JHJH 

23-mer = two turns 12-mer = one turn Molecular explanation of the rule Intervening DNA of any length 23 V DJ7 9

23-mer 12-mer Loop of intervening DNA is excised Heptamers and nonamers align back-to-back The shape generated by the RSS’s acts as a target for recombinases V1 V2 V3V4 V8 V7 V6 V5 V9 DJ V1 DJ V2 V3 V4 V8 V7 V6 V5 V9 An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the rule) Molecular explanation of the rule

23-mer 12-mer V1 DJ V2 V3 V4 V8 V7 V6 V5 V CONSEQUENCES OF RECOMBINATION Generation of P-nucleotides

23-mer 12-mer Loop of intervening DNA is excised V1 DJ V2 V3 V4 V8 V7 V6 V5 V Terminal deoxynucleotidyl Transferase (TdT) Generation of N-nucleotides

V DJ TCGACGTTATAT AGCTGCAATATA Junctional Diversity TTTTT Germline-encoded nucleotides Palindromic (P) nucleotides - not in the germline Non-template (N) encoded nucleotides - not in the germline Creates an essentially random sequence between the V region, D region and J region in heavy chains and the V region and J region in light chains

How does somatic gene rearrangement (recombination) work? 1.How is an infinite diversity of specificity generated from finite amounts of DNA? Combinatorial diversity 2.How do V region find J regions and why don’t they join to C regions? rule 3.How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity Junctional diversity (P- and N- nucleotides, see above)