Immunology - Lecture 7

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Questions and Answers

What proteins compose the V(D)J recombinase?

Artemis and TdT

RAG1 and RAG2 (correct)

DNA-PK and KU70

Ligase IV and XRCC4

In which types of cells is the RAG protein expressed?

Developing B and T cells (correct)

Mature neutrophils

All immune cells

Proliferating normal cells

What is the role of Artemis in V(D)J recombination?

Phosphorylates RAG proteins

Adds random nucleotides to DNA ends

Opens hairpins at coding junctions (correct)

Ligates the processed ends

Which enzyme participates in the nonhomologous end joining (NHEJ) repair process?

DNA-dependent protein kinase (DNA-PK) Signup and view all the answers

What is a consequence of the introduction of P- and N-nucleotides during recombination?

Diversification at the junctions between gene segments Signup and view all the answers

What sequences are formed when DNA hairpins are ligated?

Signal joints Signup and view all the answers

Which proteins are involved in the recruitment of DNA-PK during the end joining process?

KU70 and KU80 Signup and view all the answers

What does terminal deoxynucleotidyl transferase (TdT) specifically do during V(D)J recombination?

Adds nucleotides to broken DNA ends Signup and view all the answers

What regions are primarily responsible for determining the combinatorial diversity of antibodies?

V, D, and J segments Signup and view all the answers

How many possible unique Vκ regions can be generated based on the provided combinations?

1,086,750 Signup and view all the answers

What does the 12/23 Rule pertain to in the context of V(D)J recombination?

The recognition of RSS by V(D)J recombinase Signup and view all the answers

What is the primary function of the lymphocyte-specific enzyme during V(D)J recombination?

To create double-stranded breaks Signup and view all the answers

What is the total number of gene segments mentioned?

149 Signup and view all the answers

In which step of V(D)J recombination are selected coding segments brought together?

Synapsis Signup and view all the answers

What is the importance of flanking DNA sequences in the rearrangement of gene segments?

They guide the recombination process. Signup and view all the answers

How many possible V heavy chains can be produced based on the data presented?

6210 Signup and view all the answers

What is the primary purpose of gene rearrangement in lymphocyte development?

To randomly generate a diverse repertoire of receptors Signup and view all the answers

Which region of the Ig receptor is formed by sequences from both V and J gene segments?

CDR3 Signup and view all the answers

How are variable regions of immunoglobulin chains encoded?

By individual gene segments that are rearranged Signup and view all the answers

What is the role of the D segments in the immunoglobulin heavy chain locus?

To provide additional diversity between V and J segments Signup and view all the answers

Which component is NOT involved in the process of generating high-affinity B-cell clones?

Transcription of germline genes Signup and view all the answers

What does the term 'clonal selection' refer to in B-cell development?

The process of selecting cells with high affinity for specific antigens Signup and view all the answers

What does the 'germline organization' of immunoglobulin gene loci refer to?

The arrangement of V, D, and J segments before rearrangement Signup and view all the answers

Which of the following best describes the CDR3 region of the immunoglobulin receptor?

Crucial for binding to specific antigens Signup and view all the answers

What role do P- and N-nucleotides play in B-cell development?

They create unique markers for tracking B-cell clones. Signup and view all the answers

What outcome results from differential splicing of a long primary mRNA transcript in B cells?

Generation of IgM and IgD. Signup and view all the answers

What is the significance of the 12/23 rule in TCR gene rearrangement?

It specifies recombination signal sequences in the genome. Signup and view all the answers

Which component is removed through alternative splicing to create secreted immunoglobulins?

Transmembrane domains. Signup and view all the answers

How do deficiencies in RAG-1 or RAG-2 affect B and T cell populations?

They lead to a lack of B cells and T cells. Signup and view all the answers

What is a primary mechanism by which B cells generate receptor diversity?

Somatic recombination of gene segments. Signup and view all the answers

What distinguishes the surface expression of IgM and IgD on mature B cells?

They undergo differential splicing of the same pre-mRNA. Signup and view all the answers

What is the result of exonuclease trimming in the process of junctional diversity?

It generates P- and N-nucleotide insertions. Signup and view all the answers

Study Notes

Generation of Lymphocyte Antigen Receptors

Primary Ig and TCR gene rearrangement and expression is the focus of this lecture.
The goal of lymphocyte development is to randomly generate a large repertoire of antigen receptors and then select for the useful clone.
The CDR3 region of the antigen receptor is formed by the joining of two or more individual gene segments during lymphocyte development.
The variable regions of the receptor chains are not directly encoded as a complete immunoglobulin domain by a single DNA segment.
Three separate loci encode all of the Ig H chains, the Ig L chains, and the Ig L chains.
- Each locus contains a cluster of V gene segments, several J segments and in the case of the Ig H chain, additional D segments.
- The C (constant) region genes are at the 3’ end of J segments.

V regions are constructed from gene segments

V = variable
D = diversity
J = joining
VJ and VDJ combinations are possible.

Combinatorial diversity

Light chains and Heavy chains are each made up of V, D, and J segments.
Kappa and Lambda light chains can be generated.
The possible number of unique V and V light chains can be calculated by multiplying the number of available V, D, and J segments for each type of light chain.
- Possible V light chains = 35x5 = 175
- Possible V light chains = 30x5 = 150
The number of possible unique V heavy chains can be calculated similarly; this is a much larger number.
- Possible V heavy chains = 45x23x6 = 6210
The total number of possible unique V and V regions can be calculated by multiplying the number of available V, D, and J segments for each type of light chain.
- Possible unique V regions = 175x6210 = 1,086,750
- Possible unique V regions = 150x6210 = 931,500
Theoretical Combinatorial Diversity = 2,018,250

Rearrangement of V, D, and J gene segments

The rearrangement of V, D, and J gene segments is guided by flanking DNA sequences.

The 12/23 Rule

V gene segments are joined by recombination.
Recombination of V and J exons occurs by deletion of intervening DNA as well as ligation of the V and J segments.

Enzymatic steps in V(D)J Recombination

Synapsis: Two distant selected coding segments and their adjacent RSSs are brought together.
Cleavage: The V(D)J recombinase creates double-stranded breaks at RSS-coding sequence junctions.
- The V(D)J recombinase is composed of RAG1 and RAG2.
- RAG genes are expressed only in developing B and T cells.
Hairpin Opening and End Processing: Hairpins are opened at coding junctions and nucleotides can be added or removed to increase diversification.
- Artemis is an endonuclease that opens up the hairpins.
- Terminal deoxynucleotidyl transferase (TdT) adds nucleotides to broken DNA ends.
Joining: The broken coding ends and signal ends are brought together and ligated.
- KU70 and KU80 bind to the breaks and recruit DNA-PK.
- DNA-PK phosphorylates and activates Artemis.
- Ligation of the processed broken ends is mediated by DNA ligase IV and XRCC4.

Diversity of antigen receptor genes

The diversity of antigen receptor genes is generated by:
- Combinatorial diversity: The combination of different V, D, and J segments.
- Junctional diversity: The introduction of P- and N- nucleotides at the junctions between gene segments.

The introduction of P– and N- nucleotides diversifies the joints between gene segments

P nucleotides are added as a result of hairpin formation during the recombination process.
N nucleotides are added by TdT, a lymphocyte-specific enzyme.
P and N nucleotides create unique markers that are useful for tracking individual B-cell clones in studies like somatic hypermutation.

IgM and IgD are derived from the same pre-mRNA transcript

B cells expressing IgM and IgD have not undergone class switching.
The primary mRNA transcript is differentially spliced, allowing expression of both IgM and IgD.

Transmembrane and secreted forms of immunoglobulin are generated from different heavy-chain mRNA transcripts

Alternative splicing of the heavy-chain mRNA transcripts determines whether the immunoglobulin is expressed as a transmembrane or secreted form.

TCR Diversity

The germline organization of the human T-cell receptor  and  loci resembles that of Ig loci.
Both  and  chains undergo gene rearrangement.

TCR - and -chain gene rearrangement and expression

The process of TCR gene rearrangement is similar to that of Ig gene rearrangement.
The 12/23 rule applies to TCR gene segments as well.

Recombination signal sequences flank TCR gene segments

The 12/23 rule describes how recombination signal sequences (RSSs) flank TCR gene segments.

Checkpoint Questions

Somatic recombination in T cells and B cells are similar in that both processes involve the rearrangement of gene segments to create diverse antigen receptors. However, the specific gene segments and the resulting antigen receptor structures differ between T cells and B cells.
The transmembrane domain of the immunoglobulin heavy chain is removed by alternative splicing to produce soluble, secreted immunoglobulins.
RAG-1 and RAG-2 are essential for V(D)J recombination, which is the process of creating diverse antigen receptors in B and T cells. A deficiency in either RAG protein leads to severe combined immunodeficiency (SCID) because the immune system is unable to generate functional B and T cells.
The V domain of the heavy chain and light chain genes are broken up into V, D, and J segments (heavy chain) or V and J segments (light chain) that are rearranged during lymphocyte development to create a unique variable region.
Heavy chain synthesis starts with the initial rearrangement of D and J segments, followed by recombination of the V segment with the rearranged DJ segment. This creates the complete VDJ region of the heavy chain. Then, the constant region genes are joined to the VDJ region.
Light chain synthesis involves the rearrangement of V and J segments, followed by the joining of the constant region gene. This creates the complete VLJ region of the light chain.
B cells generate receptor diversity through combinatorial diversity and junctional diversity. Combinatorial diversity arises from the random selection of V, D, and J segments during gene rearrangement. Junctional diversity is generated by the addition of P and N nucleotides at the junctions between gene segments during recombination.

Assigned Readings

Chapter 5
5-1, 5-2, 5-3, 5-4, 5-5, 5-7, 5-8

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Description

This quiz covers the generation of lymphocyte antigen receptors, focusing on primary immunoglobulin and TCR gene rearrangement. It explores the complex processes involved in the development of a vast repertoire of antigen receptors, emphasizing the importance of the CDR3 region and the various gene segments involved. Test your understanding of the variable and constant regions in immunology.