(L21) IMED2001 - The Development of Immune Repertoires

The Development of Immune Repertoires

The Development of Immune Repertoires

Body Defences, Lecture 21

IMED2001

Dr. Erika Bosio

Recommended reading: Chapter 4, Basic Immunology 6th Edition, Abbas et al

Antibody

antigen-binding site

Fab

Fc

carbohydrate

T-cell receptor

antigen-binding site

α chain

β chain

variable region (V)

constant region (C)

transmembrane region

cytoplasmic tail

T cell

DIAGRAM ON SLIDE 1

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Extra notes to help with understanding:

The lecturer introduced the lecture as focusing on development of immune repertoires: how the body develops the many different B cells and T cells needed to recognise antigens. The immune repertoire means the collection of immune cell clones that can deal with different antigens.

Learning Outcomes for this lecture

1. Understand the germline configuration of TCR and immunoglobulin genes and how this segmented organisation is responsible for repertoire diversity.

2. Demonstrate a basic understanding of the process of somatic recombination required to generate functional antibodies and TCR genes.

3. Demonstrate an understanding of the contribution of combinatorial diversity and junctional diversity to the final repertoires of T and B cells in each individual.

4. Discuss and describe the processes of B and T cell development - noting similarities and differences.

5. Understand and describe the processes of lymphocyte selection to ensure cells with complete and functional receptors are maintained, while any self-reactive lymphocytes are removed and eliminated.

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Extra notes to help with understanding:

The lecturer said the learning outcomes are there for revision: students should return to them and make sure they can tick each one off when studying.

Lecture overview

Lecture overview

• The focus of the upcoming lectures is now the ADAPTIVE immune response;

• Dominated by B cells and T cells and their diverse responses

• Humoral and Cellular immunity

• Specific antigen recognition is central to all aspects of adaptive immunity;

• Development of progenitors to become effector cells (mature T and B cells)

• Triggering effector cell function

• Antigen recognition is achieved by a multitude of B cell receptors and T cell receptors

• Detect external stimuli

• Trigger responses

Antibody

antigen-binding site

Fab

Fc

carbohydrate

T-cell receptor

antigen-binding site

α chain

β chain

variable region (V)

constant region (C)

transmembrane region

cytoplasmic tail

T cell

DIAGRAM ON SLIDE 3

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Extra notes to help with understanding:

The lecturer explained that antigens are foreign substances perceived by the body as foreign. They can include viruses, bacteria, lipids, carbohydrates, sugars and other materials. Antigen processing and MHC presentation make antigens visible to the adaptive immune system. B cells drive humoral immune responses, meaning soluble immunity such as antibodies in blood and lymph. T cells drive cell-mediated or cellular immunity.

The B cell and T cell receptors

The B cell and T cell receptors

• Principal function: to detect external stimuli and trigger responses within the cells receiving the signal.

• These receptors are clonally distributed;

• Each lymphocyte clone is specific for a distinct antigen, and has a UNIQUE receptor

• This means the number of clones we have is ENORMOUS

• This collection of clones (with distinct T cell receptors, or antibodies (B cell receptors)) is referred to as the immune repertoire

Antigen X

Clonal selection

Anti-X antibody

DIAGRAM ON SLIDE 4

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Extra notes to help with understanding:

The lecturer explained that antibody molecules can be bound to the surface of a B cell, making them B cell receptors, or they can be soluble in blood. T cell receptors are always bound to the surface of T cells. If a cell labelled X recognises antigen X, it proliferates to produce lots of copies of clone X. Clonally distributed means all cells in a clone have the same receptor and therefore the same antigen specificity. Each person's immune repertoire is different, but people can still respond to the same antigen by recognising different parts of it.

But HOW do we generate so many different T and B cell receptors?

Our bodies create literally hundreds of millions of TCRs and antibodies. How is this possible if we only have 20-25,000 protein coding genes in our genome??

But HOW do we generate so many different T and B cell receptors?

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Extra notes to help with understanding:

The lecturer emphasised the problem: the body has far more possible B cell and T cell clones than there are protein-coding genes. Individual genes cannot encode every possible receptor, so another mechanism is required to generate receptor diversity.

Thinking about lymphocyte maturation

Thinking about lymphocyte maturation

• The process generates a massive number of cells, each with different receptors, and then selects those with functional/useful receptors (and the rest die)

• This has to be independent of antigen exposure, because recognition of antigen depends on those receptors already being there!

Common lymphoid progenitor

Pro-B/T cells

Pre-B/T antigen receptor expression

Immature B/T cell: expresses complete antigen receptor

Weak antigen recognition

Mature B/T cell

Positive selection

Strong antigen recognition

Negative selection

Failure to express preantigen receptor; cell death

Failure to express antigen receptor; cell death

DIAGRAM ON SLIDE 6

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Extra notes to help with understanding:

The lecturer described maturation as beginning from a common lymphoid progenitor that differentiates into precursor B cells or T cells. Cells proliferate and progress through pro-B/T and pre-B/T stages. Cells that fail developmental checkpoints die by apoptosis. The process occurs independently of antigen exposure because the receptors need to exist before antigen can be recognised. For T cells, much of this process occurs before birth in a sterile environment.

The antigen receptor genes are "special"

The antigen receptor genes are "special"

• The TCR and Immunoglobulin genes are inherited in a unique organization, called the Germline Configuration, that makes it possible to generate a massive number of different T cell receptors and antibodies.

• In all cells, except mature T and B cells, the receptor genes are fragmented and can't be expressed

• They are not present as single complete genes - they are encoded as families of gene segments

• Each set of segments contains alternative versions of parts of the receptor.

• These individual gene segments need to be re-arranged to assemble a functional gene.

Ig H chain locus (chromosome 14)

L

V (n≈45)

D (n≈23)

J

Cμ

Cδ

Cγ3

Cγ1

Cα1

Cγ2

Cγ4

Cε

Cα2

Extracellular domain

Transmembrane and cytoplasmic domains

Ig κ chain locus (chromosome 2)

L

V (n≈35)

J

C

Ig λ chain locus (chromosome 22)

L

V (n≈30)

J

Cλ1

Cλ2

Cλ3

Cλ4

Cλ5

Cλ6

Cλ7

TCR β chain locus (chromosome 7)

L

V (n≈48)

D1

J1

C1

D2

J2

C2

TCR α chain locus (chromosome 14)

L

V (n≈45)

J (n≈50)

C

DIAGRAM ON SLIDE 7

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Extra notes to help with understanding:

The lecturer explained that antigen receptor genes are inherited as segments rather than complete classical genes. For immunoglobulin heavy chains, these include V, D, J and C segments. V refers to variable, D to diversity, J to joining, and C to constant regions. Random recombination of these segments creates a new exon that forms the variable part of the receptor. These loci exist in every cell, but only B and T cells use the relevant transcription factors and chromatin accessibility to rearrange and express them.

Somatic recombination and expression of antigen receptor genes

Somatic recombination and expression of antigen receptor genes

Germline DNA at Ig H locus

5′

V1

Vn

D1-Dn

J1-6

Cμ

3′

Somatic recombination (D-J joining) in two B cell clones

Recombined DNA in two B cell clones

V1

Vn

D1J1

Cμ

V1

Vn

D3J2

Cμ

Somatic recombination (V-DJ joining) in two B cell clones

Recombined DNA in two B cell clones

V1D1J1

Cμ

VnD3J2

Cμ

Transcription

Primary RNA transcript

V1D1J1

Cμ

VnD3J2

Cμ

RNA processing (splicing)

Messenger RNA (mRNA)

AAA

Translation

Ig μ chains in two B cell clones

V

Cμ

CDR3

V

Cμ

CDR3

DIAGRAM ON SLIDE 8

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Extra notes to help with understanding:

The lecturer described somatic recombination as the process that joins V, D and J segments to create a unique variable region. In immunoglobulin heavy-chain development, D joins to J first, then V joins to the DJ segment. Once VDJ recombination is complete, transcription and RNA splicing generate a readable gene product, usually first with the μ constant region, producing IgM.

T-cell receptor diversity is generated by GENE REARRANGEMENT

T-cell receptor diversity is generated by GENE REARRANGEMENT

• The T cell receptor gene segments are less complicated than the Ig gene segments

• α chain similar to antibody light chains - V and J segments

• β chain similar to antibody heavy chains - V, D and J segments

α-chain locus

germline DNA

Lα

Vα × 70-80

Jα × 61

Cα

chromosome 14

recombination

rearranged DNA

transcription

splicing

T-cell receptor protein

β-chain locus

germline DNA

Lβ

Vβ × 52

Dβ1

Jβ1

Cβ1

Dβ2

Jβ2

Cβ2

chromosome 7

recombination

rearranged DNA

translation

splicing

transcription

DIAGRAM ON SLIDE 9

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Extra notes to help with understanding:

The lecturer explained that TCR gene rearrangement follows the same broad principle as immunoglobulin rearrangement. The TCR β chain is most similar to the antibody heavy chain because it has V, D and J segments. The TCR α chain is most similar to the antibody light chain because it has V and J segments only.

RANDOM Somatic Recombination

RANDOM Somatic Recombination

• This process is mediated by a lymphoid-specific enzyme - VDJ recombinase - which consists of the RAG-1 and RAG-2 enzymes

• Following recognition of specific flanking sequences, the VDJ recombinases bring the segments close together, and cleaves the DNA

• The nicks are then repaired by ligases, generating a VJ, or VDJ sequence, with the intervening sequence removed as an inert loop of DNA

• The VDJ recombinase is ONLY expressed in immature T and B lymphocytes

• In B cells, only the Immunoglobulin genes are rearranged (not the TCR genes) because of the influence of B cell lineage-specific transcription factors, that enable unwinding of the Ig-gene locus, but not the TCR genes. In T cells, only the TCR locus is accessible.

animations/videos of this process are available in the LMS in this week's content folder!!

Steps of TCR gene recombination

V

D

J

Recombination activating gene products, RAG1 & RAG2 and high mobility group proteins bind to the RSS

The two RAG1/RAG2 complexes bind to each other and bring the V region adjacent to the D/J region

The recombinase complex makes single stranded nicks in the DNA, the ends of each broken strand

The nicks are "sealed" to form a hairpin structure at the end of the V and D regions and a flush double strand break at the ends of the heptamers

The recombinase complex remains associated with the break

DIAGRAM ON SLIDE 10

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Extra notes to help with understanding:

The lecturer explained that VDJ recombinase is made of RAG1 and RAG2. These enzymes recognise specific signal sequences near V, D or J segments, bring the chosen segments together, cut out the intervening DNA as a loop, and allow repair enzymes to join the coding ends. This produces combinatorial diversity because different combinations of V, D and J segments are randomly chosen.

Junctional Diversity

Junctional Diversity

• Enzymatic activity during the formation of the coding joint results in further diversity

• This area corresponds to the CDR3 or HV3 domain of the developing Immunoglobulin - a very significant part of the molecule

• Believed to increase diversity by a factor of up to 3 x 10^7!

Generation of junctional diversity

RAG complex cleaves the heptamer RSSs from the D and J gene segments to yield DNA hairpins

DNA complex opens hairpins by nicking one strand of the DNA, generating palindromic P-nucleotides

N-nucleotide additions by TdT

Pairing of strands

Unpaired nucleotides are removed by an exonuclease

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

P

N

P

DIAGRAM ON SLIDE 11

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Extra notes to help with understanding:

The lecturer explained that after the hairpin ends are opened, the enzyme TdT can add random nucleotides to the loose DNA ends. These random additions help create complementarity so the DNA strands can be joined, but the number and identity of added bases are random. This creates extra variation in the CDR3/HV3 region and is believed to increase diversity enormously, by up to about 30 million-fold.

Alternative splicing of the heavy chain gene transcript

Alternative splicing of the heavy chain gene transcript

• After rearrangement, transcription starts at the 5' end, through the V region, which is upstream of the C regions.

• It continues through the Cμ and Cδ genes and stops before the Cγ3 gene.

• This creates a transcript which contains both the IgM and IgD heavy chain genes, but only one is needed to generate a complete protein.

L

VDJ

Cμ

Cδ

Cγ3

Cγ1

Cα1

Cγ2

Cγ4

Cε

Cα2

DIAGRAM ON SLIDE 12

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Extra notes to help with understanding:

The lecturer explained that after VDJ rearrangement, transcription reads through the μ and δ constant region genes. The resulting transcript can contain both IgM and IgD constant-region information, and alternative splicing decides whether the final protein becomes IgM or IgD. The process is skewed toward IgM because μ is first in the line.

Alternative splicing generates IgM and IgD in the same B cell

Alternative splicing generates IgM and IgD in the same B cell

Expression of IgM

DNA

L

VDJ

Cμ

MC

polyum

Cδ

MC

polyδm

RNA

mRNA

AAA

protein

IgM

Expression of IgD

DNA

L

VDJ

Cμ

MC

polyum

Cδ

MC

polyδm

RNA

mRNA

AAA

protein

IgD

DIAGRAM ON SLIDE 13

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Extra notes to help with understanding:

The lecturer explained that a naive B cell can produce both IgM and IgD from the same rearranged heavy-chain locus using alternative splicing. This means the same cell can express both IgM and IgD on its surface while maintaining the same antigen specificity.

Ig and TCR Diversity

Ig and TCR Diversity

• The random recombination of V, D and J segments (COMBINATORIAL DIVERSITY) is limited by the number of V, D and J segments, and results in millions of possible antibody and TCR molecules

• The process of JUNCTIONAL DIVERSITY (which will be different in every single cells) is essentially unlimited, and massively increases the number of potential receptors (10^11-10^16!!!)

Number of variable (V) gene segments:

Immunoglobulin heavy chain: ~45

κ: 35

λ: 30

T cell receptor α: 45

T cell receptor β: 48

Number of diversity (D) gene segments:

Immunoglobulin heavy chain: 23

κ: 0

λ: 0

T cell receptor α: 0

T cell receptor β: 2

Number of joining (J) gene segments:

Immunoglobulin heavy chain: 6

κ: 5

λ: 4

T cell receptor α: 50

T cell receptor β: 12

Mechanism

Combinatorial diversity:

Number of possible V(D)J combinations

Ig: ~3 x 10^6

TCR: ~6 x 10^6

Junctional diversity:

Removal of nucleotides

Addition of nucleotides (N-region or P-nucleotides)

Total potential repertoire with junctional diversity

Ig: ~10^11

TCR: ~10^16

DIAGRAM ON SLIDE 14

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Extra notes to help with understanding:

The lecturer explained that combinatorial diversity comes from multiplying the available numbers of V, D and J segments. This alone creates millions of combinations. Junctional diversity adds random nucleotides during joining, increasing potential diversity dramatically, to roughly 10^11 antibody specificities and 10^16 TCR specificities.

How do the individual T cells and B cells mature?

OK.... So we've seen how these receptors are made

How do the individual T cells and B cells mature?

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Extra notes to help with understanding:

The lecturer transitioned from explaining how receptors are generated to asking how individual B cells and T cells mature and are selected. The key issue is that receptors may be made, but the cells still need to be tested for functionality and usefulness.

Maturation and Selection of B lymphocytes

Maturation and Selection of B lymphocytes

• B lineage committed progenitors proliferate - and become pro-B cells

• And D-J rearrangement is activated, H chain first!

• Cells that successfully make a μ heavy chain survive to become pre-B cells.

• Only a small number will make a functional μ chain because...

• Junctional diversity introduces random numbers of nucleotides, but need multiple of 3 to encode amino acids to make a protein!

• Pre-B cells express the μ protein on their surface - together with two invariant proteins

HSC

Pro-B

Large Pre-B

Small Pre-B

Immature B

Mature B

Ig H chain gene:

Germline

D to J rearranged

V to DJ rearranged

Rearranged VDJ

Rearranged VDJ

Rearranged VDJ

Ig L chain gene:

Germline

Germline

Germline

V to J rearranged

Rearranged VJ

Rearranged VJ

Ig protein:

None

None

pre-BCR

Intracellular μ

IgM

IgM, IgD

DIAGRAM ON SLIDE 16

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Extra notes to help with understanding:

The lecturer explained that B cell development begins with heavy-chain rearrangement first. A functional μ heavy chain must be made. Many cells fail because junctional diversity may disrupt the reading frame: nucleotide triplets are needed to encode amino acids, so not every rearrangement produces a sensible functional protein.

Checkpoint 1: The formation of the Pre-B-cell receptor

Checkpoint 1: The formation of the Pre-B-cell receptor

• In order to survive, the pre-B cell must make a μ-chain AND that μ-chain must be able to combine with a light-chain. But- light chains haven't been made yet!

• The pre-B cells makes a surrogate light chain (VpreB and λ5)

• Combination of the μ-chain with the surrogate light chain forms the Pre-B-cell receptor

• Enables internal signaling to stop further gene rearrangement at the heavy chain locus (allelic exclusion)

Pre-B-cell receptor:

surrogate light chain

VpreB

λ5

heavy chain

Igβ

Igα

B-cell receptor:

light chain

heavy chain

Igβ

Igα

DIAGRAM ON SLIDE 17

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Extra notes to help with understanding:

The lecturer explained that the surrogate light chain tests whether the μ heavy chain can form a usable receptor-like structure before real light chains are made. The pre-B cell receptor cannot bind antigen; its job is to test the heavy chain and signal the cell to stop heavy-chain rearrangement, proliferate, and begin light-chain rearrangement.

At the Pre-B cell stage, light chain rearrangement begins

At the Pre-B cell stage, light chain rearrangement begins

• The large pre-B cells proliferate, to yield a clone of about 100 small resting cells

• All have identical μ-chain

• No longer make the surrogate light chain (so no more pre-B-cell receptor)

• RAG genes are reactivated, and light chains begin to rearrange

• Light chain rearrangement starts with the κ locus first, then the λ locus

• There are multiple possibilities on each chromosome to make productive rearrangements!

Successive rearrangements are possible at the immunoglobulin light-chain loci

Vκ1

Vκ2

Vκ3

Jκ1-5

Cκ

First VJ recombination

Nonproductive rearrangement

Second VJ recombination

Nonproductive rearrangement

Third VJ recombination

DIAGRAM ON SLIDE 18

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Extra notes to help with understanding:

The lecturer explained that once a good heavy chain has been made, the cell proliferates so many cells share that same μ chain. RAG genes then reactivate to rearrange light chains. The κ locus is tried first, and if it fails to produce a useful protein, the λ locus can be tried. The cell has multiple chances to make a productive light chain.

Checkpoint 2: Immature B cell

• Once a light chain has been made, it associates with the heavy chain to make IgM - and a complete BCR can be made once the Igα and Igβ proteins combine.

• Igα and Igβ are the B cell signalling complex, that transmit intracellular signals upon antigen recognition by surface bound antibody

• Presence of the BCR on the surface generates signals to stop light chain rearrangement

• This ensures B cells can only make one type of light chain.

• Once the BCR is on the surface, and recombination is switched off, the cell is now an IMMATURE B CELL - CHECKPOINT 2!!

Early pro-B cell

Heavy-chain gene rearrangement

First checkpoint

Light-chain gene rearrangement

Second checkpoint

Immature B cell

VpreB

λ5

pre-B-cell receptor

Igα and Igβ

B-cell receptor

no pre-B-cell receptor

apoptosis

no B-cell receptor

apoptosis

Commits to B-cell lineage

Generates heavy-chain gene diversity in pre-B cell population

Selects for functional heavy chains

Generates light-chain gene diversity in pre-B cell population

Selects for functional light chains

Makes functional IgM

DIAGRAM ON SLIDE 19

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Extra notes to help with understanding:

The lecturer explained that once the light chain works, it pairs with the heavy chain to form IgM and a complete B cell receptor. Igα and Igβ transmit signals into the cell. Surface BCR expression tells the cell to stop rearranging light chains, ensuring the B cell makes only one type of light chain. Cells that fail checkpoints die by apoptosis.

Naïve B cells make both IgM and IgD

Naïve B cells make both IgM and IgD

• Circulating B cells that have not yet encountered antigen are known as naïve B cells.

• They have undergone V-D-J rearrangement, BUT they will not complete their development until they have been stimulated by their target antigen

• They express both IgM and IgD as the result of a process of alternative mRNA splicing of the rearranged heavy chain locus.

IgM

IgD

DIAGRAM ON SLIDE 20

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Extra notes to help with understanding:

The lecturer explained that mature naive B cells have surface IgM and IgD, but have not yet met antigen. They have undergone VDJ rearrangement, but their full development into activated antibody-producing cells depends on stimulation by their target antigen.

Selection processes now target self-reactivity

Selection processes now target self-reactivity

• The immature B cell population now contains a vast repertoire of complete antigen receptors of different specificities

• Some of these WILL bind to normal body proteins

• Successful development depends on complete protein, not on specificity of recognition

• If left unchecked, this will lead to autoimmune reactivity and disease

• These cells have 2 options - either change their specificity, or die by apoptosis

• Will discuss this in more detail in later lecture on "Tolerance"

Don't ever press the red button! That is the attack self button.

Can I press it now?

Nooooo!

Re-training autoimmune cells is never easy.

DIAGRAM ON SLIDE 21

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Extra notes to help with understanding:

The lecturer explained that because receptor specificity is generated randomly, some BCRs will inevitably recognise self proteins. At this stage, selection must target self-reactivity. Self-reactive B cells must either change their specificity or die by apoptosis, otherwise autoimmune disease could result.

When it comes to T cells...

When it comes to T cells...

• T cell progenitors migrate from the bone marrow to the thymus - where they undergo all maturation steps

• Earliest progenitors are called Double negative (DN) or pro-T cells

• These proliferate under the influence of IL-7

• β chain rearrangement is happening in the DN cell

• Lots of recombination and rearrangement happening in these cells. Let's take a look...

Stem cell

Double negative (CD4−CD8−) Pro-T cell

Pre-TCR

Double positive (CD4+CD8+) immature T cell

Weak recognition of class II MHC + peptide

APC

Positive selection

Mature CD4+ T cell

Weak recognition of class I MHC + peptide

APC

Positive selection

Mature CD8+ T cell

No recognition of MHC + peptide

Failure of positive selection (death by neglect)

Apoptosis

Strong recognition of either class I or class II MHC + peptide

Negative selection

Apoptosis

DIAGRAM ON SLIDE 22

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Extra notes to help with understanding:

The lecturer explained that T cell progenitors migrate from bone marrow to the thymus. Early T cell progenitors are double negative because they do not express CD4 or CD8. They proliferate under IL-7 and begin β-chain rearrangement. Mature T cells must become either CD4 or CD8, must recognise self MHC, and must not be strongly self-reactive.

Step 1: γ:δ or β chain re-arrangement?

Step 1: γ:δ or β chain re-arrangement?

• Commitment to either lineage is competitive and complicated!

• Re-arrangement at the γ, δ, and β loci happens at the same time - the first successful re-arrangement determines which direction the cell heads down

• Either γ:δ receptor or β chain (preT receptor)

A common double-negative T-cell progenitor gives rise to αβ and γδ T cells

CD34

uncommitted progenitor

CD2

committed double-negative T-cell progenitor

γ + δ

β, γ, and δ rearrangements

β

T-cell receptor

CD8

CD4

committed γδ T cell

uncommitted double-positive thymocyte

β chain

α, γ, and δ rearrangements

γ + δ

α + β

committed γδ T cell

T-cell receptor

committed αβ T cell

DIAGRAM ON SLIDE 23

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Extra notes to help with understanding:

The lecturer explained that commitment to γδ T cells or αβ T cells is partly determined by chance. γ, δ and β rearrangements occur at the same time. To become a γδ T cell, both γ and δ must rearrange successfully. To enter the αβ pathway, the β chain only needs to rearrange successfully, so most T cells become αβ T cells.

If the first β chain is no good, the cell can try again - because there are two D J clusters

If the first β chain is no good, the cell can try again - because there are two D J clusters

• Each thymocyte has 4 attempts at making a functional β chain

• 2 attempts on each allele

• First try with Cβ1 fragment, then with Cβ2

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Extra notes to help with understanding:

The lecturer explained that thymocytes have multiple attempts to make a functional β chain because there are two DJ clusters. Cells can try rearrangements using Cβ1 and then Cβ2, on each allele, giving four attempts overall.

So what is this PreTCR??

So what is this PreTCR??

• In α:β T cells, the β chain is rearranged first, and must be functional before the alpha chain locus can commence rearrangement.

• NOT ALL REARRANGEMENTS WILL GENERATE A FUNCTIONAL GENE PRODUCT

• How can the cell determine functionality if there's only half of the parts needed to build a TCR???

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Extra notes to help with understanding:

The lecturer explained that, as in B cell development, the cell must test whether the first receptor chain is functional before the second chain is made. For αβ T cells, the β chain must be functional before α-chain rearrangement begins.

How can you test β chain function if the α chain hasn't been made yet?

How can you test β chain function if the α chain hasn't been made yet?

• A Pre-T cell receptor is formed, using an invariant polypeptide called pTα

• The pre-TCR complex delivers intracellular signals that.... (see figure!)

• The preT cell proliferates - creating a clone of cells, all with the same β chain

• This proliferation signals the surface expression of both CD4 and CD8, generating double positive (DP) thymocytes

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Extra notes to help with understanding:

The lecturer explained that the invariant pTα chain tests whether the β chain can form a receptor-like complex. If the β chain works, the pre-TCR signals the cell to stop β-chain rearrangement, proliferate, start α-chain rearrangement, and begin expressing both CD4 and CD8, producing double positive thymocytes.

α-chain rearrangement

α-chain rearrangement

• The recombination machinery is re-activated, and targeted to the α chain locus, but not the β locus.

• Repeated attempts at α chain gene rearrangement are also possible

• Once a productive α-chain rearrangement is made, the protein is translocated into the ER to test recognition of the β chain

• Recognition of the β chain is check point 2 - provides important survival signal, and progression to positive selection.

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Extra notes to help with understanding:

The lecturer explained that after a successful β chain has been made, recombination machinery is redirected to the α-chain locus. The cell can make repeated α-chain rearrangement attempts. If the α chain recognises and pairs properly with the β chain, checkpoint 2 is passed and the cell can move toward positive selection.

What we know so far......

What we know so far......

• At the end of this first part of the T cell development process;

• DP thymocytes

• Set of rearranged TCR genes

• Functional TCR (correctly folded and potentially useful)

• Diverse population of immature cells

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Extra notes to help with understanding:

The lecturer summarised that by this stage, T cell development has produced double positive thymocytes with rearranged TCR genes and a functional TCR. These cells are still immature and require selection to determine whether they are useful and safe.

Generation of Functional T and B cells depend on the process of RAG-mediated recombination

Generation of Functional T and B cells depend on the process of RAG-mediated recombination

• What if the RAG enzymes didn't work or weren't made in an individual?? (eg. Affected by genetic mutation)

• Answer: No T or B cells

• SEVERE IMMUNODEFICIENCY - called "SCID"

Severe Combined Immunodeficiency

• Bone marrow transplant needed to restore function

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Extra notes to help with understanding:

The lecturer explained that dysfunctional RAG enzymes prevent VDJ recombination, so functional B cells and T cells cannot be generated. This causes severe combined immunodeficiency (SCID). It is "combined" because both B and T cell development are affected. Bone marrow transplant can restore function by providing cells capable of reconstituting the immune system.

Selection in the thymus removes autoreactive T cells - Central Tolerance

Selection in the thymus removes autoreactive T cells - Central Tolerance

• At the completion of this process, these now DOUBLE POSITIVE cells undergo further selection and maturation to become mature, single positive CD8 or CD4 T cells

• Two step process;

• first POSITIVE SELECTION in the thymic cortex

• then NEGATIVE SELECTION at the corticomedullary junction

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Extra notes to help with understanding:

The lecturer explained that central tolerance removes autoreactive T cells that arise because TCRs are created randomly. The thymus selects useful T cells and deletes harmful self-reactive cells. Positive selection occurs in the thymic cortex, while negative selection occurs as cells move toward the corticomedullary junction.

Positive Selection

Positive Selection

• Outcome: to positively select T cells that can bind to self-MHC

• Remember, there are thousands of different MHC isoforms in humans

• Only about 2% of an individual's T cells will be able to recognize their specific MHC Class I or II molecules

• Cortical epithelial cells express complexes of self-peptide and MHC (both Class I and Class II)

• In the absence of infections, these cells present self-peptides generated during the normal breakdown of the body's own proteins

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Extra notes to help with understanding:

The lecturer explained that positive selection checks whether a T cell can recognise self MHC. No binding is bad because the cell would not participate in immune responses. Very strong binding is also bad because the cell may react to self. The desirable outcome is moderate binding to self MHC-peptide complexes, which gives the cell a survival signal.

Positive Selection

Positive Selection

• Positive selection determines whether the cell will become a CD4 or CD8 T cell, depending on which MHC Class it's TCR binds to.

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Extra notes to help with understanding:

The lecturer explained that the MHC class recognised during positive selection helps determine lineage. Recognition of MHC class II drives CD4 T cell development, while recognition of MHC class I drives CD8 T cell development. At the end of this process, the cell becomes single positive rather than double positive.

Negative Selection

Negative Selection

• Single positive cells moving from the cortex to the medulla, next come into contact with DCs and macrophages at the corticomedullary junction

• Negative selection is dependent on the strength of TCR/MHC binding with these cells

• Strong/tight binding - cells die by apoptosis

• Low/moderate binding - survival signal

• Outcome - removal of autoreactive cells, generation of a repertoire of single positive mature T cells, ready to fight infection!

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Extra notes to help with understanding:

The lecturer explained that during negative selection, single positive T cells interact with dendritic cells and macrophages presenting self peptide-MHC. Strong binding suggests recognition of self antigen and leads to apoptosis. Low or moderate binding provides survival signals. The outcome is a mature single positive T cell repertoire ready to fight infection while reducing autoreactivity.