Antigen receptors_ Diversity

Question 1

Between the heavy and the light chain, which one undergoes VDJ rerrangement?

Answer 1

The heavy chain only. Light chain only has VJ rearrangement

Question 2

Describe the VDJ rearrangement process in the diagram below

Answer 2

**note that VDJ recombination is referring only to the HEAVY CHAIN** (the light chain, lambda or kappa, doesn’t have a D gene)

As the B cell matures, it splices out all the genes but the ones it wants to keep (so for the V’s, everyone gets spliced out except for the one it wants to keep, and the same for D, J and C genes)

In mature B cell DNA, there’ll be the V gene that the cell wants to keep and the D gene that the cell wants to keep. It hangs on to the J gene for the time being until its ready to transcribe mRNA

In mRNA, you’ll have the selected V gene, D gene, J gene and still the different genes for the C chain

In the heavy chain, the B cell chooses from 50 V genes, 25 D genes, and around 6 J genes (all found on chromosome 14)

**note that the light chain only has V and J choices only, no D. Why? I’m guessing this makes sense because the region that confers effector function, which is the heavy chain, actually has the effector function (partly) determined by the D chain (diversity, because different Abs subtypes will respond to different things)**

**note also that V, D, and J are the genes that encode for the CONSTANT portion of the heavy chain, which makes up the Fc portion of the antibody, which you will recall is also the effector portion**

**note also that the constant regions for IgM and D are the first to be synthesized

(note that you have D and J coming together first, then you have V joining to a DJ segment)

Question 3

The ___ chain of the B cell is analogous to the alpha chain of the TCR, and the __- chain is analogous to the beta chain of the TCR

Answer 3

The heavy chain of the B cell is analogous to the beta chain of the TCR, and the light chain is analogous to the alpha chain of the TCR

Question 4

In VDJ rearrangement, what is the significance of the recombination signal sequences?

What is it made up of?

Answer 4

The recombination signal sequences sit between the genes and basically flank the regions where DNA needs to be rearranged

The RSS is made up of a heptamer, either 12 or 23 nucleotides which correspond to one or two turns of the dna double helix (respectively, and a nonamer

Question 5

The 2 enzymes that clip the RSS’s in VDJ rearrangement are ___ and ___

Answer 5

RAG 1 and RAG 2

Question 6

What are the functions of RAG1 and RAG2?

T/F: Both enzymes are expressed only in developing T and B cells

Answer 6

RAG1 – endonuclease that binds to and cleaves the RSS’s

RAG2 – activated by histone binding to the -vely charged chromatin; also attracted to the RSS and makes a dimer with RAG1 on RSS’s

Truth. Both are required for T and B cell development

Question 7

Mutations in RAG 1 and 2 can lead to which two immune deficiency conditions?

Answer 7

(if you scid on road with snow and you got bad omen, you gon crash)

SCID

Omenn’s syndrome

Question 8

During VDJ rearrangement, the cut pieces of dna are added to the enzyme ___, which randomly adds N nucleotides, helping to generate more diversity and chaging the antigen specificity

Answer 8

The cut end of the dna is going to be added on to the enzyme Terminal Deoxynucleotide Transferase (TdT). This enzyme randomly adds and removes N nucleotides (N as in any nucleotide), which helps to add more diversity even among cells that have the same VDJ genes, and changes the antigen specificity

Question 9

Upon addition of the nucleotides by TdT, the cut pieces of DNA are joined via a process known as ___

Answer 9

Non homologous end joining

Question 10

In non homologous end joining, ___ sits on the broken ends and serves almost like a signal for DNA PKcs to come find the broken dna

Answer 10

Ku70/86

Question 11

What is the role of DNA PKcs in putting the broken dna back together?

Answer 11

DNA Protein Kinase binds each end of the broken dna (recruited by Ku70/86)

DNA Protein Kinase then recruits Artemis

Question 12

___ is an exonuclease that clips off the broken single stranded ends of the dna

Answer 12

DNA Protein Kinase then recruits Artemis, an exonuclease that cuts off the single stranded ends

Question 13

What is the role of DNA ligase?

Answer 13

DNA Ligase IV then ligates the ends together (also with the help of XLF-XRCC4)

Question 14

Deficiencies in any of the NHEJ proteins results in ___

Answer 14

Deficiencies in any of the NHEJ proteins results in:

Increased susceptibility to ionizing radiation

Immunodeficiency due to impaired VDJ rerrangement >> no B and T cell development

Question 15

When the unwanted DNA segments are looped out in VDJ rearrangement, they form ___, which are used in bone marrow transplantation as markers of the age of the cells in the bone marrow

Answer 15

When the unwanted DNA segments are looped out in VDJ rearrangement, they form replication excision cirles, which are used in bone marrow transplantation as markers of the age of the cells in the bone marrow

Question 16

What is the difference between combinatorial and junctional diversity?

Answer 16

Combinatorial diversity refers to the diversity generated just by the random reassortment of VDJ genes

Junctional diversity refers to the diversity generated by the various processes within VDJ rearrangement that contribute ADDITIONAL diversity like when TdT adds random nucleotides such that even two cells expressing the same VDJ arrangement will be slightly different in their nucleotide sequence, and the NHEJ process which is error-prone so that contributes diversity as well

Question 17

Describe allelic exclusion

Answer 17

Allelic exclusion: literally you exclude the expression of one of the two alleles for a gene. In this case, we’re talking about each heavy chain, light chain (both kappa and lambda) or beta and alpha chain having two alleles and when we’re making the receptors for these, only one of the alleles is expressed

Question 18

How is allelic exclusion utilized in the process of making a functional heavy/light or alpha/beta chain?

Answer 18

After you’ve made the B cell heavy chain or the T cell beta chain, you need to check if the chain can link up with a functional light chain or alpha chain

You pair the heavy chain (or beta chain) with a VpreB surrogate light chain (or a pre-T-alpha surrogate for the beta chain). If the two can pair up and the resulting receptor is functional, then a signal is sent back to the cell to proliferate. If the receptor is not functional, then the cell tries to rearrange (i.e. uses the OTHER ALLELE for that chain and sees if that one works instead)

**note that this is applicable to the light chain too**

Question 19

Omenn syndrome is a type of SCID caused by mutations in RAg 1 or 2.

A mutation in which RAG enzyme affects RSS DNA binding or catalytic activity?

A mutation in which RAG enzyme affects chromatin accessibility of VDJ recombinase?

Answer 19

RAG-1 mutations affect RSS DNA binding or catalytic activity

RAG-2 mutations affect chromatin accessibility of VDJ recombinase

Question 20

Mutations in Artemis/DNA ligase can cause combined SCID and what type of sensitivity?

Answer 20

SCID + increased radiation sensitivity

Question 21

VDJ recombination can also lead to chromosome translocations and ___ malignancies

Answer 21

VDJ recombination can also lead to chromosome translocations and lymphoid malignancies

**if the translocation involves Myc genes, this can result in malignancy**