Lectures 5 and 6, IG/TCR Variability Devo Flashcards by Dave Ed

In B lymphocytes, what is the name of the chains that undergo rearrangement?
What specific parts of each chain rearrange?

Heavy and light chains

Light: V J
Heavy: D J followed by V DJ

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What is the name of the process whereby germline DNA rearranges?

Somatic recombination

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What occurs after somatic recombination and prior to translation?

Splicing

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Describe the path from HSC to mature B cell

HSC -> Pro-B cell -> Pre-B cell -> Immature B cell -> Mature B cell

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*How do we know, during B cell development, that the cell has become a Pre-B cell?

Mew heavy chain is expressed

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*How do we know, during B cell development, that the cell has become an immature B cell?

Surface IgM expressed

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How do we know, during B cell development, that the cell has become an mature B cell?

Surface IgM and IgD expressed

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*When, during B cell development, does heavy chain (D-J and V-DJ) rearrangement occur?

During pro-B cell stage

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*When, during B cell development, does light chain (V-J) rearrangement occur?

During pre-B cell stage

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What is allelic exclusion? Why does it occur?

How does it to occur?

Only 1 of the 2 alleles (chromosomes) is rearranged (you only want 1 heavy and 1 light chain to be rearranged)

As a result of allelic exclusion, all the antigen receptors on an individual lymphocyte will have the same AA sequence in the variable domain of the heavy chain
RAG

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What protein cuts during light and heavy-chain gene rearrangement?

RAG-1/2

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*How does RAG know where to cut?

Binds at a conserved sequence known as Recombination Signal Sequence (RSS)

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During light chain gene rearrangement, where would RSS’s be found?

Downstream of V’s, upstream of J’s

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During Ig gene rearrangement, what are the names of the fragments that form via RAG in B cells? T cells?

BRECs and TRECs
(B cell Recombination Excision Circle)
(T cell Recombination Excision Circle)

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How could BRECs and TRECs be important clinically?

BRECs and TRECs indicate that new cells are being made. They dilute out over many cell divisions
- Important info to know for marrow transplants

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*What mechanism allows for creation of Ig’s w/same variable domains that can be either secreted or membrane bound?

Alternative RNA splicing

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Where is the most downstream place that RNA cleavage occurs, and is crucial for whether an IG is secreted or membrane-bound?

Poly-A tail

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What’s a solid estimate of the amount of different epitopes we need?

~10 million (10^7)

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What are the 3 methods by which such a large AB repertoire is generated?

Combinatorial V(D)J gene joining
N nucleotide addition by TdT (Terminal deoxynucleotidyl Transferase)
Combinatorial association of H and L chains

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How does TdT act to enhance AB diversity?

How many NT’s does it typically add?

After excision by RAG, rather than simple ligation TdT adds NTs b/w V(D)J segments randomly.
- Typically adds 1-20 NTs

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How could TdT NT addition lead to problems?

If NTs aren’t added in units of 3, you would have a frameshift.

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What happens to the cells if V(D)J rearrangements are nonfunctional?

They die

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*What if V(D)J rearrangements react w/a multivalent self molecule?
What if they react w/ soluble self molecules?

They apoptose or undergo receptor editing

- They migrate to periphery and become anergic

What happens to an immature B cell that does not react to self?

Migrates to periphery and become a mature B cell

Where does receptor editing take place?

Bone marrow

What is receptor editing? | What part of the B cell does it affect?

If B cell reacts to multivalent self molecule but doesn't apoptose (because it hasn't left marrow?), RAG is reactivated and light chain rearrangement continues - Likely will no longer react to self

*What are the names of the TCR chains? Compare them to BCRs/IGs.

Alpha and beta chains - Alpha similar to light chain (V + J) - Beta similar to heavy chain (V(D)J)

Do T cells express Ig's on their surface like B cells?

Can T cells express Ig's in membrane-bound and in secreted form?

In the HSC, what is the state of the genes considered?

Germline (no rearrangements)

T/F? A single pentameric IgG will express both kappa and lambda light chains?

False

Do gamma-delta and alpha-beta T cells share rearranged TCR genes or not?

No--Both T cells, but have separate gene segments

T/F, B and T cell leukemias express multiple TCR/BCR?

False--they are monoclonal

*When only the heavy chain is expressed on a B cell's surface, what stage is the cell at? A type of IgM is still seen on the surface- What is this called? What is the Ig/receptor called?

- Pre-B cell stage - A surrogate light chain is expressed - Pre-B cell receptor

*What is the purpose of having a pre-B cell receptor?

To shut off RAG, thus preventing further heavy chain gene rearrangement (important for allelic exclusion)

What are the names of the 2 different light chains that can be made? Which is made first? What is the typical ratio in humans?

- Kappa and lambda - Kappa is rearranged 1st - 65:35 kappa:lambda

*How could the V(D)J rearranged heavy chain segment possibly lose it's D?

If the V(D)J rearrangement leads to a (multivalent) self-reactive AB, receptor editing occurs if the cell survives. During receptor editing, a new V J rearrangement can occur that would delete out the D segments in a BREC, forming only a V-J segment. (but doesn't only the light chain undergo receptor editing?)

If a heterozygote w/1 parent that passed on x-heavy chain and 1 parent that passed on y-heavy chain genes, which heavy chain would be expressed in a single Ig? Would all Ig's have x or y, or could some be x and some be y, or would most be mixed x and y?

- Either all x or all y heavy chain would be expressed in each Ig due to allelic exclusion - Across all Ig's, there could be some that are all x and some that are all y, but no mixed Ig's.

What is myeloma?

A type of cancer that begins in the bone marrow. It is a cancer of plasma cells (a type of B cell) - Multiple myeloma is most common form

What is leukemia? | What's a major side effect?

A malignant progressive disease in which the bone marrow and other blood-forming organs produce increased numbers of immature or abnormal leukocytes. - These suppress the production of normal blood cells, leading to anemia and other symptoms.

What mechanism is responsible for an Ig's w/ the same variable domains to have different constant domains? (e.g. IgA vs. IgD)

Alternative splicing (at RNA level)

Once AB is already formed (e.g. IgM or IgD in B cell), how could these Ig's become others such as IgG, IgE, or IgA? (Overall name of the process)

Isotype switching

Where does isotype switching occur?

Secondary lymphoid tissue

What protein is involved in isotype switching? What is this analogous to in B cell gene rearrangements? What is the site that this protein recognizes? What is this analogous to in B cell gene rearrangements?

AID (analogous to RAG) Switch regions (analogous to RSS)

If an IgM undergoes isotype switching to become an IgG, could it ever go back to IgM? Could it go on to become an IgA/IgE?

No (that segment has already been excised/used) | Yes

Do IgM (produced first) and IgD on the surface of the same B cell have the same or different V regions?

Same (just different C regions)

How can μ and δ heavy chains arise from the same transcript of a B cell?

Mew and delta (heavy) chains with the same Vh domain result from alternative splicing of primary transcripts (nuclear RNA).

How do you calculate the maximum possible number of combinations (VDJ rearrangements) in a B cell?

#V x #D (if present) x #J | product

What % of T cells are gamma-delta (vs. alpha-beta)?

Only ~5%

Identify the approximate number of heavy chain V, D and J gene segments in the B cell germline. Identify the approximate number of light chain V and J gene segments in the B cell germline

Heavy: ~40V, ~25D, ~6J | Light (kappa and lambda): ~70V, 4-5J

Immature B lymphocytes are called immature B lymphocytes because why?

They do not proliferate and differentiate in response to antigens.

The RSS are highly conserved stretches of __-__ nucleotides separated by nonconserved ___ or ___ nucleotide spacers.

7-9 | 12 or 23

T/F: In pre-B cells, both heavy and light chain genes are rearranged.

False- Light chain genes are in germline configuration (recall: pre-TCR)

Do T cells do receptor editing?

Which has a larger total diversity- TCRs or IG's?

TCRs (10^18 vs. 10^13)

T/F: At the immature B cell stage, once a B cell expresses a complete heavy or light chain, it cannot produce another heavy or light chain containing a different V region.

True. (but it can do receptor editing of the same chain)