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Immunology (M1) > Antibody and T cell receptor diversity > Flashcards

Antibody and T cell receptor diversity Flashcards

1
Q

Antibody structure: chains

A

2 identical heavy chains, 2 identical light chains

Each chain has Ig domains ~110 AA in length, held by disulfide bond

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2
Q

IgG Ab structure

A

L chains: 2 Ig domains
H chains: 4 Ig domains

disulfide bonds between heavy & light, heavy & heavy

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3
Q

Ig Variable regions

A

Sequence varies among antibodies; 1 Ig domain/chain

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4
Q

Ig Constant regions

A

Sequence is the same among different antibodies; 3+ Ig domains/chain

CH: other effector functions
CL: no effector functions

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5
Q

Light vs Heavy chain regions

A

Light: 1 V + 1 C per chain
Heavy: 1 V + 3 or more V per chain

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6
Q

Antigen binding site

A

1 VL + 1 VH

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7
Q

Hypervariable regions/CDRs

A

Greatest diversity in the H and L variable regions; ~10 AA long

Also called complemetarity-determining regions (CDRs) b/c bind to antigen

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8
Q

CDR location on protein

A

extend away from protein to contact antigen

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9
Q

Antigen binding pockets

A

Two per antigen, consist of VH and VL chain

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10
Q

H chain isotypes

A

IgA, D, E, G, M

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11
Q

L chain isotypes

A

πœ… and πœ†

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12
Q

Secreted vs. Membrane-Associated Antibodies

A

Differ at C-terminal tail; Membrane = Cπœ‡4 followed by: cytoplasmic tail (3 aa, CY) + hydrophobic transmembrane region (26 aa, TM)
Secreted = Cπœ‡4 followed by: hydrophilic tail; uses poly(A) cleavage site before TM-CY exons, not included in primary transcript

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13
Q

In the membrane-associated form, all isotypes are ____

A

Monomeric

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14
Q

T Cell Receptor structure/function

A

Recognizes antigen in context of MHC

two chains: 𝛼𝛽 or 𝛾𝛿
each chain: V and C region

CDRs in variable region that bind antigens

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15
Q

Mechanisms of Ab diversity

A
  1. Multiple VH/L genes
  2. Combinatorial association of different V, D, J gene segments
  3. Junctional diversity by nucleotide addition
  4. Combinatorial association of VH/L regions
  5. Somatic hypermutation
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16
Q

Ab gene rearrangement: Heavy chain

A

gene: V, D, J, C
CDR1/2 derived from VH segment
CDR3 derived from DH and JH segments

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17
Q

Ab gene rearrangement: Light chain

A

gene: V, J, C
CDR1/2 derived from VL segment
CDR3 derived from JL segments

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18
Q

Ab gene organization

A

Gene pieces assembled during lymphocyte development (3 loci, different chromosomes)
Heavy: (V, D, J), C
Light: (V, J), C

19
Q

Leader Sequence

A

Each V segment (V/D/J) has L (leader) sequence, codes for N-terminal signal peptide (targets protein to endoplasmic reticulum)

20
Q

VDJ recombination steps

A
  1. chromatin opens, giving recomb enzymes access to genes
  2. 2 gene segments brought together, double strand break
  3. Nucs added/removed, ends ligated
  4. RNA splicing of introns
  5. each B cell has different combo of V gene segments/diversity due to addition of N/P nucs
21
Q

Recombination signals

A

RSSs - mediate recombination; 7 nt stretch (CACAGTG) separate by 12 or 23 spacer (1-2 turns, bring heptamers together), followed by concerved AT-rich stretch of 9 nt

12 bp spacer can only join to 23 bp segment

22
Q

VDJ recombination: deletion vs inversion

A

Deletion: heptamer synapses join together, gene is cut, creates a circle of intervening DNA

Inversion: when RSSs are 3’ of Jπœ… gene, intervening DNA is not removed, stays until RNA splicing

23
Q

Rearrangement and Transcription

A

Rearrangement brings enhancers close to promoter allowing transcription to occur

24
Q

Non-homologous end joining purpose

A

Repairs double strand breaks by joining the two broken ends together and ligating them without template

uses small ss region of homology to enhance repair

25
Q

Non-homologous end joining process

A
  1. Ku70/80 binds to break, recruits DNA-dependent protein kinase (DNA-PK)
  2. DNA-PK phosphorylates and activates Artemis endo-exonuclease
  3. Artemis endonuclease trims unpaired DNA
  4. Ligase complex ligates the DNA
26
Q

VDJ joining: Synapsis

A
  1. Chromatin opens
  2. Chromosome looping
  3. Recombination enzymes (RAG1 and 2) form complex, recognize RSS sequences
  4. RAG2 binds to hypermethylation in chromatin, recruits/activated RAG1
27
Q

VDJ joining: Cleavage

A

RAG1 nicks one strand, ss DNA covalently forms hairpin with other strand

28
Q

VDJ joining: Hair pin opening, end processing, joining

A
  1. Ku70/80 binds to breaks, recruits DNA-PK (repair enzyme, activates Artemis)
  2. Artemis endonuclease opens hairpins
  3. DNA polymerase fills in nucleotides (P)
  4. terminal transferase (TdT) adds nucs to broken DNA ends (N)
  5. DNA ligase joins ends together
29
Q

Junctional diversity

A

P (hairpin cleavage + DNA polymerase) and N (TdT) nucleotide addition; CDR3 = greatest diversity

30
Q

Most important region for Ab binding specificity

A

CDR3

31
Q

Allelic Exclusion

A

Each B cell only makes one Ab; Only one chromosome undergoes rearragement

Other is excluded (heterochromatized/methylated)

32
Q

Light chain isotype exclusion

A

If the πœ… locus undergoes a productive rearrangement, it inhibits πœ† rearrangement

33
Q

Affinity Maturation/Class Switching

A

During immune response, B cells produce Abs that progressively have a higher affinity for antigen

Abs switch from IgM to G, A, E

34
Q

Switch Regions

A

1-10 kb long GC-rich regions between J and C gene segments

Upstream of S region: I exon & I region promotor

Cytokine stimulation triggers transcription (I, S, CH)

35
Q

Mechanism of Class Switching

A
  1. CD40 reacts with B cells, induces AID
  2. AID removes amino group from cytosines –> deaminated uracil, ultimately leading to double strand breaks at πœ‡ switch region and downstream S region
  3. πœ‡ switch region joins downstream CH switch region, intervening DNA lost
  4. RNA splicing yields mRNA with new CH region
36
Q

Activation-Induced Cytidine Deaminase Mechanism

A
  1. AID removes amino group from cytosines –> deaminated uracil
    a) modify GC-rich tetranuc motifs, increase pol II stalling
    b) pol II stalling recruits AID
    c) RNA-DNA hybrid allows ss-DNA R loop, targeted by AID
  2. Uracil N-glycosylase (UNG) removes U residues –> abasic
  3. Ape I endonuclease nicks each abasic site
  4. Exosome degrades RNA from D loop, AID + Ape convert + nick
    RESULT: ds break in two S regions
37
Q

Affinity Increase Cause

A

Somatic mutations in V region, enhance affinity for antigen (somatic hypermutation – mutation rate 1000x higher that other genes)

Require helper T cells and CD40:CD40L signaling

Mutations cluster in CDRs, cause increased afffinity

38
Q

Somatic Mutation Mechanism

A

In V region, AID converts C -> U at AGCT hotspots, cause 3 changes:

  1. DNA replication, U -> T
  2. UNG excises bases and repairs with error prone system
  3. Mismatch repair enzymes (MSH2/6, Exol) remove U nucs, replace surrounding DNA with error prone DNA pol
39
Q

T Cell Receptor Diversity

A 
𝛼𝛽 and 𝛾𝛿 pairs
𝛽, 𝛿 – VDJ; 𝛼, 𝛾 – VJ
 𝛽, 𝛿 – VDJ; 𝛼, 𝛾 – VJ
𝛽: 50 V, 2 D, and 12 J segments
𝛼: 45 V and 50 J segments
𝛾, 𝛿: much smaller - 7 V genes total

V regions have CDR1-3 with CDR3 the most diverse
𝛽, 𝛾: 2 C regions
𝛼, 𝛿: single C region

V(D)J joining and P/T diversity like antibody genes

40
Q

Severe Combined Immunodeficiency (SCID)

A

Severe mutations or deficiencies in recombination enzymes (RAG1, RAG2, Artemis, DNA-PK, DNA Ligase 4) result in a complete loss of B cells and T cells

41
Q

Omenn Syndrome

A

Hypomorphic mutations with reduced (not absent) function of RAG and Artemis genes

less severe than SCID

42
Q

Hyper IgM Syndrome

A

Genetic defects in AID or UNG are defective in class switching and affinity maturation

All IgM

Can be caused by defects in CD40

43
Q

Lymphoid Tumors and Translocations

A

B and T cell tumors commonly have translocations of oncogenes into antibody loci

AID ds breaks oncogenes near S sites, join in CSR break –> over expression of oncogene, uncontrolled growth

Can have RAG-RAG or RAG-AID translocations

Immunology (M1) (11 decks)

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