Antigen Recognition Genetics

Describe the difference in antigen recognition between B/T Cells

B vs T:

• Antigen Types:

  • B:

    • Outside the cell

    • linear peptides or complex 3D; or any kind of molecules

  • T:

    • Primarily protein antigens

    • linear peptides

• Presentation:

  • T cells must be presented an antigen by APCs on MHC

Describe the structures of antibodies polypeptides

• Describe the different regions

• How is an antibody isotype class determined?

• Define:

  • Isotypes

  • Allotypes

  • Idiotypes

• What is the difference between a B Cell Receptor (BCR) and the secreted Ig (sIg)

Regions:

• Variable Region:

  • higher degree of variation in amino acid sequence

    • [] @ N terminal end

• Constant Region:

  • limited degree of variation btw dif. antibodies

• Hinge region:

  • Flexible

  • relatively unstructured portion in the middle

• Antigen binding site:

  • formed by V regions of L and H chains

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Determination of Ig(antibody) isotypes

• Dependent on Heavy chain C regions

  • α, δ, γ: 3 domains

  • μ, ε: 4 C domains

    • extra domain = elongated hinge region

• For Light Chains:

  • Either Kappa or Lambda in all types

  • No funcitonal differences

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Definitions:

• Isotypes:

  • same thing as antibody classes (IgM, IgD, IgG, IgE, and IgA)

• Allotypes:

  • genetically determined differences in antibodies between people

• Idiotypes

  • Antibodies of different specificity found within the same individual due to the diversity of the Ig V region

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BCR vs sIg:

• same specificity

• Difference:

  • one is membrane-bound; other = secreted.

• BCR signaling = mediated by two small proteins w/ long cytoplasmic tails: Iga and Igb

  • AKA CD79a and b.

Describe the V domain

• Framework region

• Hypervariable region

V Domain:

• Framework Regions:

  • Flanks the hypervariable regions

  • Much less variable

• Hypervariable regions:

  • Regions w/in V domain where it is super variable

  • AKA CDRs (Complementarity-determining regions)

    • b/c provide a binding surface that is complementary to that of antigen.

Describe the Characteristics of B Cells:

• Antigen Specificity

• Antigen Repertoire

B Cells Characteristics:

• Antigen Specificity:

  • each B cell is specific for only one epitope of an antigen.

• Antibody Repertoire:

  • number of different antibodies/B cells recognizing different epitopes that an individual can generate.

Describe how the mechanisms to generate B cell Diversity

• List the Different Domains

• Describe Rearrangement/Recombination Mechanism:

  • What is RSS? Components?

  • what is the 12/23 rule

  • What does RAG do?

• Describe Junctional Diversity

  • 3 components?

  • Resolving

  • End result

The Different Domains:

• V = variable region gene segment

• D = diversity region gene segment (only in Ig heavy chain genes)

• J = joining region gene segment

• C = constant region gene segment

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Rearrangement/Recombination Mechanism:

• RSS: (Recombination Signal Sequences)

  • Special DNA motifs that directs recombination site

  • Components:

    • Heptamer(7) region:

      • Always adj. to Variable Region’s Coding Sequence

    • Nonamer(9) Region:

    • Spacer Region:

      • Separates Heptamer and Nonamer regions

      • Either 12 or 23 bps

• 12/23 Rule:

  • RSS w/ 12bp spacer interacts only w/ RSS w/ 23bp spacers

    • ensure gene segments are joined in correct order

• RAG1 & RAG2 (Recombinase Activating Genes 1 & 2)

  • Mediate V-(D)-J Joining

    • Rags attach to the RSS of the V/J regions

    • They join together and Cut the DNA

    • The VJ segments gets joined together (Coding Joint) → Retained in genome

      • everything originally between the V/J gets jointed together (Signal Joint)→ floats away

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Junctional Diversity

• 3 Components:

  • Junctional flexibility:

    • RAGs induced double-strand cuts → creates DNA Hairpin

  • P-nucleotide addition:

    • Another enzyme (Artemis, act. by DNA-PK) cuts near end of RSS → allows the Non-coding Strand (bottom strand) to join the Coding Strand (top strand)

  • N-nucleotide addition:

    • TdT (Terminal deoxy-nucleotidyl transferase) (lymphocyte specific) → adds random # of nucleotides @ end of RSS (after P addition)

• Resolving the Junction:

  • Pairing of Strands

  • Unpaired nucleotides = removed by exonuclease

  • Gaps are filled by DNA synthesis and ligation to form coding joint

• End Result:

  • Some = non-productive

    • accidentally created “stop” codons or introducing frameshifts in the J region

  • Some = Productive

    • will generate increased diversity in the third hypervariable region (CDR3).

Describe what happens if there is Absent or Defective RAG Proteins

Absent or Defective RAG Proteins:

• SCID

  • T and B cells are absent

• Omen syndrome

  • Chronic inflammation from auto-reactive T cells

• Treatment: Bone Marrow Transplant

1. Describe Allelic Exclusion

2. Describe Clonal Selection

Allelic Exclusion

• After productive rearrangement of one heavy chain and one light chain gene → recomb. of other allele does not proceed.

  • result in a B cell becoming monospecific to only one antigen

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Clonal Selection

• B cell binds its cognate antigen → signaling effect → proliferation to generate enough effector cells to fight

1. Describe the naive B cell

   • which Ig does it express? how?

   • Difference between Secreted vs membrane Ig

2. Describe class Switchin

   • Occurs when?

   • Describe the difference in the primary vs secondary immune response; Difference between the two Igs?

   • Describe the genetic contents for the Ig heavy chain

   • Mechanism?

3. Describe Somatic Hypermutation:

   • Mediated by?

   • Mech

Naive B Cells:

• Express both Surface IgM & IgD

  • Expression of both isotype accomplished by alternative mRNA splicing

    • ***NOTE: The constant region for IgM and IgD is close together; so after rearrangement; that’s why they’re both expressed together***

• Secreted vs membrane Ig:

  • same selectivity; only genetic difference is the C-terminus

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Class Switching

• Occurs AFTER Antigen Exposure

• Primary vs Secondary Response:

  • Primary: Mostly IgM

  • Secondary: Mostly IgG

  • Difference:

    • IgG = same specificity/heavy chain VDJ and light chain VJ gene segments as IgM

    • ONLY THE CONSTANT REGION HAS CHANGED!!

• Genetics of Ig heavy Chain

  • has Multiple Constant Domain Regions (determining different types of heavy chains)

    • ORDER matters in isotype switching

• Mech:

  • Mediated by AID (Activation-Induced cytidine deaminase)

  • AID binds to 2 Switch regions

    • Areas located before each specific constant region

    • One = orgiinal; the other = the new isotype it wants to become

  • The aid brings the Switch region together →

    • cut everything in the middle

    • only the new constant region remains (along its switch region; the switch region of the Original is also gone)

  • After Switching = no switching back (b/c that region is lost)

    • (you can still switch if you have some other constant regions after the New one though)

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Somatic Hypermutation:

• Mediated also by AID

• Mech: Introducing Point Mutation ONLY IN V REGION → alters Ab affinity

  • C + AID → U + repair → A,C,G, or T

Describe the CD3 -T Cell Receptor (TCR) Complex

• Function

• Deficiencies:

  • CD3γ or CD3ε

  • CD3δ / CD3ζ

• Function

  • CD3 complex proteins transmit signals to cell after TCR binds peptide/MHC

• Deficiencies:

  • CD3γ or CD3ε deficiency

    • Low TCR numbers

    • Impaired signal transduction

  • CD3δ / CD3ζ deficiency

    • T cells absent

    • Normal B cells

    • NK cells: impaired cytolytic function

1. What are teh two types of T Cell Receptor

2. Describe γδ TCells

   1. Percentage?

   2. Location?

   3. Interact w/

   4. Recognize/Triggered by?

   5. γδ vs αβ?

   6. γδ Chains?

Two types:

• γδ

• αβ

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γδ Tcells:

• 1-5% of circulating T cells

• Dominant in gut mucosa, epithelial tissues and skin epidermis

• Interact with nonclassical MHC class 1-like CD1 receptors

• May recognize microbial phospholipid antigens

• May be triggered by “danger” signals

• γδ TCRs = less diversity than αβ TCRs

  • Fewer V gene segments

• Most γδ T cells express same γδ TCR chains.

  • Restricted TCR = pattern recognition receptor

Draw out how an γδ or αβ cell can be made

Describe the Mechanism to make either an γδ or αβ cell

Mech:

• Thymocytes Try to Correctly Rearrange a b-Chain (Similar to the Ig Heavy Chain) (V joined by D/J)

• Rearrangement of the a-Chain Gene Only Occurs in Pre-T cells

• IF FAIL → γδ = default

• If SUCCESS → no going back to γδ

Compare and Contrast T and B cell receptors

• Similarites

• T vs B:

  • membrane/Secreted

  • Ag Binding

• Both Has:

  • Variable and constant regions

  • Complementary determining regions (CDRs)

  • Gene rearrangements → variability

  • Associated membrane-bound signaling molecules

• T vs B:

  • Membrane/Secreted:

    • T: Membrane-bound (receptor) only

    • B: Membrane-bound or secreted

  • Ag binding:

    • T: No further changes

      • Only recognize antigen in MHCs

    • B: Class Switching/Somatic hypermutation