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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. Heavy Chain. Light Chain.

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slide1
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
Human Immunoglobulin Loci

Heavy Chain

Light Chain

Abbas & Lichtman’s Basic Immunology 4-9

heavy chain synthesis
Heavy Chain Synthesis

J1-n

D1-n

5’

3’

Cm

Cd

Cg

Ca

Ce

V1

V2

  • Variable (VH) segments
  • Diversity (DH) segments
  • Joining (JH) segments
  • Conserved (CH) segments

Vn

Germline DNA

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

heavy chain synthesis1
Heavy Chain Synthesis

J1-n

D1-n

Germline DNA

5’

3’

Cm

Cd

Cg

Ca

Ce

V1

V2

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

Vn

Rearranged B cell DNA

5’

3’

Cm

Cd

Cg

Ca

V1

V2

Ce

Vn

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

heavy chain synthesis2
Heavy Chain Synthesis

Rearranged B cell DNA

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

5’

3’

Cm

Cd

Cg

Ca

V1

V2

Ce

Vn

Rearranged B cell DNA

5’

3’

Cm

Cd

Cg

V2

Ca

Ce

V1

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

heavy chain synthesis3
Heavy Chain Synthesis

Rearranged B cell DNA

5’

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

3’

Cm

Cd

Cg

V2

Ca

Ce

V1

Primary RNA transcript

3’

5’

Cm

Cd

V2

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

heavy chain synthesis4
Heavy Chain Synthesis

Primary RNA transcript

3’

5’

Cm

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

Cd

V2

mRNA transcript

3’

5’

Cm

V2

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

heavy chain synthesis5
Heavy Chain Synthesis

mRNA transcript

3’

5’

Cm

V2

  • The mRNA is translated by ribosomes into m chain.

m heavy chain polypeptide

NH2

Cm

COOH

V2

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

light chain synthesis
Light Chain Synthesis

J1-n

  • Variable (Vk) segments
  • Joining (Jk) segments
  • Conserved (Ck) segment
  • 2 isotypes l (40%) and k (60%)

3’

5’

Ck

V1

V2

V3

Vn

Germline DNA

light chain synthesis1

J1-n

3’

5’

Ck

V1

V2

V3

Vn

Light Chain Synthesis

Germline DNA

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

Rearranged B cell DNA

3’

5’

Ck

V3

V1

V2

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

light chain synthesis2
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.

Rearranged B cell DNA

3’

5’

Ck

V3

V1

V2

Primary RNA transcript

3’

5’

Ck

V3

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

light chain synthesis3

Primary RNA transcript

3’

5’

Ck

V3

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

mRNA transcript

3’

5’

V3

Ck

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

light chain synthesis4
Light Chain Synthesis

mRNA transcript

  • The mRNA is translated by ribosomes into k chains.
  • Combines with heavy chain to make IgM.

3’

5’

V3

Ck

k chain polypeptide

NH2

COOH

Ck

V3

light chain synthesis5
Light Chain Synthesis

m heavy chain polypeptide

NH2

Cm

COOH

V2

k chain polypeptide

NH2

COOH

Ck

V3

IgM

Abbas & Lichtman’s Basic Immunology 4-12

mrna splicing
mRNA Splicing

IgD mRNA

3’

5’

V2

Cd

Primary heavy chain RNA transcript

3’

5’

Cm

Cd

V2

IgM mRNA

5’

3’

Cm

V2

Abbas & Lichtman’s Basic Immunology 4-12

ig class change
Ig Class Change
  • 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.

Abbas & Lichtman’s Basic Immunology 7-10

ig class change1
Ig Class Change
  • Involves a switch sequence upstream of the constant region gene segments.
  • Recombination occurs between the Sμ recombination region and a downstream switch region (Sg).
  • 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.

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

ig class change2
Ig Class Change
  • Class change occurs upon B cell activation and maturation.
  • Requires signals from ____________________________ (CD3+, CD4+, CD8-).
    • Binding of receptors

(CD40 ligand)

    • Cytokines

Abbas & Lichtman’s Basic Immunology 7-9

mechanisms of antibody diversity
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
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

G

C

A

C

T

G

G

G

T

G

T

A

  • Recombined DNA

Ser

Arg

Trp

Tyr

  • Protein
Junctional Diversity (N- region diversity)
  • J segment
  • V segment
  • 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).

A

C

T

G

T

G

C

G

G

A

T

G

  • Germ line DNA
junctional diversity n region diversity1

G

G

C

T

G

G

T

T

G

A

C

  • Recombined DNA

Val

Arg

Trp

  • Protein
Junctional Diversity (N- region diversity)
  • J segment
  • V segment
  • 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).

A

C

T

G

T

G

C

G

G

A

T

G

  • Germ line DNA
junctional diversity n region diversity2

G

T

C

C

A

G

G

C

T

T

G

G

A

T

  • Recombined DNA

Leu

Arg

Trp

Val

  • Protein
Junctional Diversity (N- region diversity)

T

C

  • J segment
  • V segment
  • 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).

A

C

T

G

T

G

C

G

G

A

T

G

  • Germ line DNA
surface vs secreted

sIgM

Surface vs. Secreted

Soluble IgM mRNA

3’

5’

Primary RNA transcript

Cm

Ts

V2

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

3’

5’

Cm

Ts

Tm

V2

mIgM

  • Poly A
  • Poly A

Membrane IgM mRNA

3’

5’

Cm

Ts

Tm

V2

membrane bound ig
Membrane Bound Ig

B cell receptor (BCR) complex

  • IgM or IgD
  • Associated with a heterodimer of Iga and Igb.
  • Activation of the BCR results in activation of Iga and Igb leading to cytoplasmic signaling and B cell activation
  • Iga
  • Iga
  • Igb
  • Igb
antibody kinetics
Antibody Kinetics
  • __________________________ is the tendency of antibody to form stable complexes with an antigen
  • +
  • Ag
antibody kinetics1
Antibody Kinetics
  • _________________________ is the strength in which a multivalent antibody binds a multivalent antigen.
  • Ag
  • Y
  • Y
  • Y
  • Y
  • Y
  • Y
  • IgG
  • IgM
t cell receptor
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
    • a and b genes are expressed in a majority of peripheral T cells.
    • g and d are expressed on a minority of thymic and peripheral T cells (10%).
  • Binds to the peptide-MHC complex.
t cell receptor1
T Cell Receptor

Variable domain

  • 2 a chains
  • 2 b chains
  • Associated with CD3
    • 2 e chains
    • 1 d chain
    • 1 g chain
  • 2 z chains

Conserved domain

CD3

Adapted from Roitt’s Immunology 4-6

t cell receptor loci
T-Cell Receptor Loci

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

t cell receptor diversification
T Cell Receptor Diversification

J1-n

3’

5’

Ca

V1

V2

Vn

Germline a DNA

J21-n

J11-n

D2

D1

3’

5’

Cb2

Cb1

V1

V2

Vn

Germline b DNA

  • Variable (VH) segments
  • Diversity (DH) segments
  • Joining (JH) segments
  • Conserved (CH) segments
t cell receptor diversification1
T Cell Receptor Diversification

J1-n

3’

5’

Ca

V1

V2

Vn

Germline a DNA

J21-n

J11-n

D2

D1

3’

5’

Cb2

Cb1

V1

V2

Vn

Germline b DNA

  • Recombination between V, D, and J gene segments.
    • a and g chains contain V and J gene segments.
    • b and d chains contain V, D, and J gene segments.
    • Similar to Ig.
comparison of tcr and ig
Comparison of TCR and Ig
  • 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

Abbas & Lichtman’s

Basic Immunology 4-1

antigen recognition
Antigen Recognition
  • 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

Abbas & Lichtman’s

Basic Immunology 4-7

next time
Next Time
  • T cell maturation
  • Cell-mediated immune responses (CMI)
  • Humoral immune responses
  • B cell activation
  • Readings: Abbas & Lichtman, Chapters 5 & 6
objectives
Objectives
  • Describe the structure and synthesis of the various immunoglobulin Heavy and Light chains, and T-cell receptors.
    • V, D, J, C Segments
  • Describe the process of immunoglobulin class change.
  • Describe the mechanisms of antibody diversity.
    • Combinitorial vs Junctional
    • Surface vs Secreted
  • Know the difference between affinity and avidity.
  • Compare and contrast immunoglobulins and T-cell receptors.