Aims Gene rearrangement and class switching of B- cell Igs. T cell receptors- What are they and...

Aims

• Gene rearrangement and class switching of B-cell Igs.

• T cell receptors- What are they and where do they fit into immunology?– Gene rearrangement to generate diversity

• Readings: Abbas & Lichtman, Chapters 4 & 7

Human Immunoglobulin Loci

Abbas & Lichtman’s Basic Immunology 4-9

Heavy Chain

Light Chain

Heavy Chain Synthesis

• Variable (VH) segments

• Diversity (DH) segments

• Joining (JH) segments

• Conserved (CH) segments

V1 V2 Vn5’ 3’Germline DNA

J1-nD1-n

CCCCC

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Heavy Chain Synthesis

• Intervening DNA between the D and J segments is looped and cut out.

5’ 3’

V1 V2 Vn5’ 3’

Germline DNAJ1-nD1-n

CCCCC

CCCCCV1 V2 Vn

Rearranged B cell DNA

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Heavy Chain Synthesis

• Intervening DNA between the V and D segments is looped and cut out.

5’ 3’CCCCCV1 V2 Vn

5’ 3’CCCCCV1V2

Rearranged B cell DNA

Rearranged B cell DNA

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Heavy Chain Synthesis

• The gene is transcribed resulting in a primary RNA containing the VHDHJH segment, the remaining J segments and only the C and C segments.

5’ 3’CCCCCV1V2

5’ 3’CCV2

Rearranged B cell DNA

Primary RNA transcript

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Heavy Chain Synthesis

• The transcript is processed and the introns and C sequences are removed. The resulting mRNA contains the C segment and will code for an IgM.

5’ 3’CCV2

Primary RNA transcript

5’ 3’CV2

mRNA transcript

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Heavy Chain Synthesis

• The mRNA is translated by ribosomes into chain.

mRNA transcript

NH2 COOHCV2

heavy chain polypeptide

5’ 3’CV2

Adapted from Abbas & Lichtman’s Basic Immunology 4-12

Light Chain Synthesis

• Variable (Vk) segments

• Joining (Jk) segments

• Conserved (Ck) segment

• 2 isotypes (40%) and (60%)

V1 V2 V3 Vn C

J1-n

3’5’Germline DNA

Light Chain Synthesis

• Intervening DNA between the V and J segments is looped and cut out.

V1 V2 V3 Vn C

J1-n

3’5’

Germline DNA

V1 V2V3 C 3’5’

Rearranged B cell DNA

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Light Chain Synthesis

• The B cell transcribes a segment of DNA into a primary RNA transcript that contains a long intervening sequence of J segments and introns.

V3 C 3’5’Primary RNA transcript

V1 V2V3 C 3’5’

Rearranged B cell DNA

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Light Chain Synthesis

• This RNA transcript is processed into mRNA by splicing the exons together.

V3 C 3’5’Primary RNA transcript

V3 C 3’5’mRNA transcript

Similar to Abbas & Lichtman’s Basic Immunology 4-10

Light Chain Synthesis

• The mRNA is translated by ribosomes into k chains.

• Combines with heavy chain to make IgM.

V3 C 3’5’mRNA transcript

V3 C COOHNH2

chain polypeptide

Light Chain Synthesis

V3 C COOHNH2

chain polypeptide

NH2 COOHCV2

heavy chain polypeptide

IgM

Abbas & Lichtman’s Basic Immunology 4-12

mRNA Splicing

Primary heavy chain RNA transcript

5’ 3’CCV2

5’ 3’CV2

5’ 3’CV2

IgD mRNA

IgM mRNA

Abbas & Lichtman’s Basic Immunology 4-12

Ig Class Change

Abbas & Lichtman’s Basic Immunology 7-10

• All classes of immunoglobulin use the same set of variable region genes (ie V-D-J).

• All that changes is the constant region of the heavy chain.

Ig Class Change

Adapted from Abbas & Lichtman’s Basic Immunology 7-10

• Involves a switch sequence upstream of the constant region gene segments.

• Recombination occurs between the Sμ recombination region and a downstream switch region (S).

• The intervening region containing genes for IgM and IgD in this instance is looped out and then cut, with deletion of the intervening regions and joining of the two switch regions.

Ig Class Change

Abbas & Lichtman’s Basic Immunology 7-9

• Class change occurs upon B cell activation and maturation.

• Requires signals from ____________________________ (CD3+, CD4+, CD8-).– Binding of receptors

(CD40 ligand)

– Cytokines

Mechanisms of Antibody Diversity

• Combinatorial diversity– Multiple germ line V genes recombining with J and D

segments.

• Junctional diversity– Imprecision in V-J and V-D-J recombinations (N-

region diversity)– N-nucleotide additions (N-region diversity)

• Note addition is done without a template.

– Somatic point mutation

• Assorted heavy and light chains

Combinatorial Diversity

• Possible combinations of V-D-J and V-J germ line gene segments.

Abbas & Lichtman’s Basic Immunology 4-11

Junctional Diversity (N- region diversity)

• Changes in nucleotide sequence at junctions of V, D, and J segments.

• Two types of changes– Exonuclease removal of

nucleotides.

– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).

C

V segment J segment

C G G TGA T AT G G

Germ line DNA

Arg Ser TyrTrp

Protein

C G G TGA T AT G G

Recombined DNA

C

Junctional Diversity (N- region diversity)

• Changes in nucleotide sequence at junctions of V, D, and J segments.

• Two types of changes– Exonuclease removal of

nucleotides.

– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).

C

V segment J segment

C G G TGA T AT G G

Germ line DNA

Arg ValTrp

Protein

C G G TTG A CT G G

Recombined DNA

Junctional Diversity (N- region diversity)

• Changes in nucleotide sequence at junctions of V, D, and J segments.

• Two types of changes– Exonuclease removal of

nucleotides.

– Random addition of nucleotides by terminal deoxyribonucleotidyl transferase (TdT).

C

V segment J segment

C G G TGA T AT G G

Germ line DNA

Arg Leu ValTrp

Protein

C T

C G G ATC G TT G G

Recombined DNA

T A C

Surface vs. Secreted

• Which poly A site is transcribed determines whether the protein is surface or secreted.

Primary RNA transcript

5’ 3’CV2

5’ 3’CV2

Soluble IgM mRNA

sIgM

Ts

Poly A

Tm

Poly A

Ts

5’ 3’CV2

Membrane IgM mRNA

Ts Tm

mIgM

Membrane Bound Ig

• IgM or IgD • Associated with a

heterodimer of Ig and Ig.

• Activation of the BCR results in activation of Ig and Ig leading to cytoplasmic signaling and B cell activation

B cell receptor (BCR) complex

Ig Ig

Ig Ig

Antibody Kinetics

• __________________________ is the tendency of antibody to form stable complexes with an antigen

+ Ag

Antibody Kinetics

• _________________________ is the strength in which a multivalent antibody binds a multivalent antigen.

Y Y

Y Y Y

IgM

Y

IgG

Ag

T Cell Receptor

• Present on a majority of T cells both CD4+ and CD8+.

• Heterodimeric molecule.– Linked by a disulfide bond.

• Generated by 4 sets of genes– and genes are expressed in a majority of

peripheral T cells.

– and are expressed on a minority of thymic and peripheral T cells (10%).

• Binds to the peptide-MHC complex.

T Cell Receptor

• 2 chains• 2 chains

• Associated with CD3– 2 chains– 1 chain– 1 chain

• 2 chains

Adapted from Roitt’s Immunology 4-6

Variable domain

Conserved domain

CD3

T-Cell Receptor Loci

Adapted from Abbas & Lichtman’s Basic Immunology 4-9

T Cell Receptor Diversification

V1 V2 Vn5’ 3’

Germline DNA

J1-n

C

V1 V2 Vn5’ 3’

J11-nD1

C

Germline DNA

J21-n

C

D2

•Variable (VH) segments

•Diversity (DH) segments

•Joining (JH) segments

•Conserved (CH) segments

T Cell Receptor Diversification

V1 V2 Vn5’ 3’

Germline DNA

J1-n

C

V1 V2 Vn5’ 3’

J11-nD1

C

Germline DNA

J21-n

C

D2

•Recombination between V, D, and J gene segments.· and chains contain V and J gene segments.· and chains contain V, D, and J gene segments.·Similar to Ig.

Comparison of TCR and Ig

Abbas & Lichtman’s Basic Immunology 4-1

• Ig binds to proteins, polysaccharides, Lipids, and nucleic acids, while TCR only binds peptides in MHC.

Forms of Antigen

Recognized

Diversity

Antigen Recognition

Mediated by

Signaling functions

Mediated by

Effector functions

Mediated by

Antigen Recognition

Abbas & Lichtman’s Basic Immunology 4-7

• Ig binds antigen all by itself, while TCR needs CD4 or CD8 to simultaneously bind.

Antigen Binding

Region

Structure of antigen

recognized

Affinity of antigen

binding

On-rate and Off-rate

Accessory molecules

Involved in binding

Next Time

• T cell maturation

• Cell-mediated immune responses (CMI)

• Humoral immune responses

• B cell activation

• Readings: Abbas & Lichtman, Chapters 5 & 6

Objectives

1. Describe the structure and synthesis of the various immunoglobulin Heavy and Light chains, and T-cell receptors.

1. V, D, J, C Segments

2. Describe the process of immunoglobulin class change.

3. Describe the mechanisms of antibody diversity.1. Combinitorial vs Junctional2. Surface vs Secreted

4. Know the difference between affinity and avidity.5. Compare and contrast immunoglobulins and T-cell

receptors.