T cell development, generations of receptor repertoire diversity

Question 1

Multipotent HSCs give rise to distinct B and T cell lineages

Describe the pathway of multipotent HSCs to B/T cell lineage

Answer 1

Multipotent HSC commits into common lymphoid progenitor

Could commit into B cell lineage

Or could commit to T cell lineage due to important signals like Notch 1, GATA3

Common progenitor that has committed to the t cell lineage can differentiate further and become mature alpha beta or gamma delta t cells.

Mature T cells are of two kinds depending on the type of T cell receptor they express, either alpha beta t cell receptor or gamma delta t cell receptor

Also some cells that branch out from lymphoid lineage early on, include ILCs- innate lymphoid cells

Accumulate into tissues

Question 2

Stages of T cell maturation (development)

Major events take place in transitional parts

What occurs during stem cell to pro-lymphocyte stage?

Pro-lymphocyte to pre-lymphocyte?

Answer 2

Growth factor mediated commitment, proliferation, initiation of antigen receptor gene rearrangement.

Selection of cells that express pre-antigen receptors.

Question 3

Stages of T cell maturation (development)

Major events take place in transitional parts

What occurs during pre-lymphocyte to immature lymphocyte stage?

Answer 3

Selection of repertoire and acquisition of functional competence

In T cells this happens in two places, early on stem cells and early multipotent progenitors will originate in the bone marrow and in the foetal liver.

Migrate to thymus

End of development in thymus they exit and go to peripheral lymphoid organ or tissue like lymph nodes and spleen

Process is not dependent on antigen but during selection and late stages antigens are important – not exogenous antigens but self-antigens

Combo of self-antigens and MHC molecules drive the process

Question 4

How does the thymus appear in a histology section?

What two areas can be seen

Answer 4

Early on in development thymus populated by precursors that commit to T cell lineage

Cortex seen in darker colour, medulla in lighter

With magnification you can see dense network of stromal cells, specifically epithelial cells and lymphocytes.

Question 5

The journey of T cells through development

Answer 5

Original precursors move early on from the bm into the thymus or from foetal liver into thymus

Specific signals such as Notch1 from the thymic stroma cause precursors to commit to T cell lineage and differentiate into early precursor of T cells

Notch signals induce the activation of transcription factor, GATA3, essential for the process of lineage commitment and development of early T cell precursors

T cell precursors undergo intense proliferation

Several waves of proliferation of lymphocytes in thymus

thymus never changes in size because over 98% of T cells in thymus die because once early precursors commit into T cell lineage and start process of development (e.g rearranging t cell receptor), may fail in production of T cell receptor or may finish in a T cell receptor that will fail selection

Selection kills many cells

Once cell passes both positive and negative selection cells can leave thymus and get in contact with APC in periphery, lymph nodes and spleen

become activated cells that can carry out the effector function – either activate macrophages or kill virally infected cells

Question 6

Successive stages in T cell development are marked by changes in surface receptors

What are the changes in surface receptors that occur?

Answer 6

1 week after arrival of precursors into the thymus progenitors commit to the T cell lineage

Express early markers of the T cell lineage (CD2 and Thy1)

Do not express any of the markers that define T cells later in development or after in the periphery (CD3, CD4, CD8)

Because of absence of CD4 and CD8 early developing T cells are called DN (double negatives)

At DN stage developing T cells (thymocytes) re-arrange the TCR locus

Stages post DN stage are characterised by expression of both CD4 and CD8 and later just one or the other

Question 7

How do early T cell comitted progenitors progress from double negative?

Answer 7

Early T cell comitted progenitors don’t express CD3,4,8 - double negative

first rearrange TCR to express CD3, then 4 and 8, large and active

proliferate vigorously

transition stage of being small and resting, expressing CD4 and CD8

undergo selection, CD4 and CD8 lineage commitment, lose one or other receptor to become fully functional CD4 positive T cell or CD8 positive T cell

exits into periphery

Question 8

Upon successful rearrangement and if selected for to be in periphery T cells express high levels of TCR

What is the TCR structure?

What domains does TCR contain?

Answer 8

TCR is a heterodimer consisting of two transmembrane peptide chains covalently linked to each other by disulphide bonds

• Beta chain
• Alpha chain

Two types of TCR, alpha-beta and gamma delta, expressed by different groups of T cells

Chain has one Ig-like variable terminal domain (V), one Ig-like constant domain (C), hydrophobic transmembrane region and a short signalling cytoplasmic region.

Question 9

What do the V regions of both alpha and beta chains contain?

Answer 9

Short stretches of amino acid sequence that is highly variable between receptors

These regions form the CDRs / complementary determining regions

3CDRs of the alpha chain and 3 of beta chain form the peptide-MHC binding site

Question 10

TCR is similar to immunoglobulin but there are specific differences

Answer 10

Ig has heavy and light chain, TCR has alpha beta or gamma delta

Number of Ig domains in TCR are 1 V and 1 C domain per chain. Ig heavy chain has more C domains.

Number of CDRs involved in antigen binding – 6 in TCR, 3hree in each one of the alpha and beta chains of TCR. Also 6 in Ig.

Signalling is not exclusively downstream to the receptor, both TCR and Ig have associated signalling molecules - CD3 and zeta chain in TCR and Ig alpha and beta in Ig

Differences in how receptors are activated – cellular activation of T cells exclusively from membrane bound peptides, no isotope switching on TCR or somatic mutations.

There is isotope switching and somatic mutations in Ig and Ig can bind to soluble antigens.

Question 11

The TCR is not alone in its function

What residues bring the chains together?

Answer 11

Cysteine residues in C regions bring the chains together.

Charged residues in the transmembrane region bind to CD3 and zeta chain to form the TCR signalling complex.

CD3 and zeta allow for the transduction of signals upon MHC-peptide binding.

Question 12

What are the features of antigens that are recognised by T cells?

Answer 12

1. Most T cells recognise peptides because recognition requires close association with MHC molecules, only peptides bind to MHC molecules.
2. Recognise linear peptides, not conformation determinants of protein antigens
   * Because linear peptides bind to clefts of MHC molecules and protein confirmation is lost during generation of these peptides
3. T cells recognise cell-associated and not soluble antigens - TCR recognise peptide-MHC comlezes, MHC molecules are membrane proteins that display stably bound peptides on cell surfaces
4. CD4+ and CD8+ T cells preferrentially recognise antigens sampled from extracellular and cytosolic pools respectively.
   * Pathways of assembly of MHC molecules ensure that class II molecules display peptides that are derived from extracellular proteins and taken up into vesicles in APCs and that class I molecules present peptides from cytosolic proteins; CD4 and CD8 bind to nonpolymorphic regions of class II and class I MHC molecules respectively.

Question 13

What is the anchor residue of MHC?

What is polymorphic residue?

Answer 13

Elements of peptide preferentially bind to TCR, other parts bind to MHC

Peptides only have few of these residues, amino acids that bind to MHC called anchor residues.

Polymorphic residues of mHC that recognise TCR, interact with TCR independent of peptide.

in development TC need to be selected to be able to bind self MHC, if can’t complete interaction they will be useless. - molecular principle of MHC restriction

Question 14

What is MHC?

Answer 14

MHC is the major histocompatibility complex

HLA Human Leukocyte Antigen – also called this

MHC class I and MHC class II

MHC class I molecules present peptide antigens derived from pathogens that replicate inside the cell, such as viruses

MHC class II molecules present peptides from pathogens and antigens that are present outside the cell taken up by endocytic vesicles of phagocytic cells

Question 15

Describe the strucure of MHC

Answer 15

Extracellular peptide binding cleft

Ig like domain

Cytoplasmic tail

MHC class II has a conserved CD4 binding site

MHC class I has a conserved CD8 binding site

Question 16

MHC molecules are polymorphic and polygenic

What does this mean?

Answer 16

Polymorphic - multiple variants of each gene within the population

Polygenic - contains several different MHC class I and class II genes

thus every individual possesses a set of MHC molecules with different ranges of peptide binding specificites

Question 17

Polymorphic residue location and MHC peptide interactions

Describe the peptide MHC interaction

Answer 17

Each MHC has one cleft that binds one peptide at the same time but can bind different peptides due to high levels of diversity.

Peptides that bind one MHC share structural features that promote binding – has preferential binding to an MHC

Acquire peptides while assembled inside the cell

Peptide-MHC interactions are saturable with low off rate, once peptide is there it can stay on top of MHC for long period of time to activate as many T cells as possible

Very small number of MHC peptide complexes can activate a T cell

MHC molecules can bind and display both foreign and self-peptides

MHC class II binds to longer peptides than class I

Question 18

Function and expression of MHC molecules

Answer 18

MHC class I molecules have conserved regions that bind to CD8 and therefore able to detect TCRs coming from CD8 positive T cells.

Peptides ontop of MHC class I molecules originate from inside the cell.

CD4 helper T cells express TCRs that bind to MHC class II molecules.

Peptides on MHC class II come from extracellular space through phagocytosis and processing within the phagolysosome pathway.

MHC class I – expressed by all cells but erythrocytes

MHC class II – expressed by antigen presenting cells

Any cells that can be infected by virus should be able to present peptides (MHC 2) to cytotoxic T cells

Question 19

Pathway of antigen processing and presentation on top of MHC class II

Answer 19

MHC class II present peptides that come from extracellular space.

Protein antigen is phagocytosed, goes into phagocytic pathway to be destroyed via enzymes and acidic environments within lysosomes

At same time the cell assembles MHC class II molecules in ER and blocks peptide binding groove via association of LI chain and CLIP

MHC class II molecule exported to the golgi

Through vesicles, fusion with lysosomes and in this process CLIP molecule is released and exchanged by a peptide that has affinity for the diverse binding groove

MHC class II molecule exported into plasma membrane for presentation onto a CD4 positive T cell

Process prevents endogenous peptides from binding to the MHC class II molecule

Question 20

What are the properties of the TCR?

Answer 20

Only one form of TCR is expressed on each T cell

Means that each T cell and its daughter cells have only one TCR and one specificity for antigen

This is a T cell clone

However, there are an infinite number of different versions of the TCR each with a unique binding site

A TCR has only one antigen binding site

A TCR is never secreted unlike Ig

Question 21

What happens after gene rearrangement?

Answer 21

Process of gene rearrangement checked

Cells that have particular specificity are selected

Once a cell is able to bind self MHC and is functional, it is exported to the periphery

Because of randomness of gene rearrangement it is likely to bind foreign antigen

Because of process of selection it is unlikely to bind to self-antigen

Once it finds foreign antigen, it proliferates and becomes an effector t cell

Question 22

Germ line configuration before rearrangement

What is germ line configuration?

Answer 22

Germ line configuration is the configuration of the inherited DNA at the TCR alpha and beta loci, and in gamma loci.

Human TCR locus is in chromosome 7, contains many gene segments, D and J encode V fragment and constant segments/C segments that form C fragment of beta chain.

Question 23

What is the alpha delta chain encoded by?

Answer 23

Alpha and delta chain is encoded by chromosome 14.

• No D segments in alpha loci
• Recombination between V, J and later C

Question 24

What is TCR gamma chain encoded by?

Answer 24

In TCR gamma chain there is similar configuration in chromosome 7 (similar to alpha delta chain)

• V beta, v alpha and v gamma to form alpha chain
• Likewise for D and j

Question 25

What mediates TCR gene segment arrangement?

Answer 25

Arranged in similar pattern to Ig gene segments and by same genes, Rag1 and Rag 2

recombination activating gene 1 and gene 2

TCR concentrate diversity in third hypervarable region CDR3

Question 26

Describe process of beta chain rearrangement

Answer 26

Start with beta chain:

Initial process of recombination mediated by RAG, joins D and J fragments

Second process of recombination joins V beta fragment with DJ fragments

Primary transcript is spliced, removes elements in the middle

Joins C fragment to VDJ

Translation will form a Beta chain

L segment, the leader sequence, tells cell where to send this polypeptide chain

Checkppoint and sucessful rearrangement of beta chain allows for initiation of the rearrangement of alpha chain.

Question 27

Describe rearrangement of alpha chain

Answer 27

Doesn’t have D fragment, has V to J event of recombination

Production of the transcript

Splicing

Formation of alpha chain

Once the two are together, the T cell can assemble a full T cell.

This process is antigen-independent. Happens in absence of antigen as processes happen in utero or early in life.

Question 28

Each T cell receptor chain is made up from different gene segments ( TCR beta chain)

Answer 28

V beta and C beta in beta chain

Fragments that cell can choose from – V,D, J, C

Process of recombination leads onto an RNA which has specific fragments and has spliced elements in between to bring constant region close

Protein is produced

Beta chain rearrangement happens first, then alpha chain

Question 29

When is TCRa chain genes rearranged?

How many times can rearrangement be attempted?

Answer 29

Do not have D gene segments and rearranged after the TCR beta chain locus.

Successive rearrangements may be attempted until a productive rearrangement has been achieved

Alpha chain can attempt more times process of rearrangement than beta chain, cell has used a lot of energy to produce beta chain

Management of alpha chain allows for more events of rearrangement until a productive rearrangement of the receptor is achieved

Question 30

What is junctional diversity?

Answer 30

During the joining of different gene segments, addition (or removal) of nucleotides may create new sequences at junctions

• Cell can change the polypeptide sequence or entire reading frame
• Giving cell receptor that’s even more diverse

Mediated by TdT terminal deoxynucleotidyl transferase

Question 31

Compare the diversity between Ig and TCR

Answer 31

TCRb doesn’t have many D segments, 2. Ig has 23 in heavy chain

N region diversification (Addition of nucleotides) between different segments in TCR

Lots of joining segments in TCRb, more than Ig

Total potential repertoire with junctional diversity (Addition or extraction of nucleotides) is 10^11 in Ig, 10^16 in TCRb and 18 for TCRa

Question 32

Gene rearrangements and checkpoints occur within particular regions of the thymus

Expression of what allows us to identify stages DN1-4

Answer 32

Various stages characterised by the absence of co receptors CD4 and CD8

T cells develop first with no expression of CD4 and CD8

Then double positives and single positives

Can be further characterised by expression of CD44 and CD25

Allows us to identify four different stages – DN1, 2,3, 4 according to acquisition and loss of CD44 or 25

Initial stages, DN1 (high in CD44, low in CD25) have TCR in the germ line configuration, haven’t started gene rearrangement

Gene rearrangement takes place between DN2 and DN3 – transition

Initial steps of rearrangement conclude with a rearranged beta chain when cells are at DN4 stage

Then begin to proliferate

Have to go through checkpoint

Checkpoint tells thymus that the cell has successfully rearranged the beta chain

Cell exports beta chain to surface with a temporary alpha chain, pre-T alpha

Question 33

What are the consequences of successful TCR beta chain rearrangement?

Answer 33

Has to signal to stromal cells in thymus around the developing T cell that it has concluded a successful B chain rearrangement

Sends B chain to surface with surrogate chain, chain that mimics alpha chain, pre-T alpha

This is the pre-t receptor

Signalling through the pre-TCR suppresses expression of the RAG genes temporarily so no more gene rearrangements can take place

This is allelic exclusion

Important as cell doesn’t want more rearrangements in the other allele corresponding to TCR

Both B and T cells only express TCR for one allele, excluding the other

Process to ensure there is allelic exclusion

Allelic exclusion ensures that only one TCR beta chain gene is expressed

events together are known as b-selection

Question 34

What happens at DN4 stage after successful signalling from PreTCR?

Answer 34

Halts further beta chain rearrangements

induces proliferation and expression of CD4 and CD8

downregulation of RAG1 and RAG2 lost, they’re re-expressed to initiate alpha chain rearrangement

cells now express CD4 and CD8