T cell development, generations of receptor repertoire diversity Flashcards
Multipotent HSCs give rise to distinct B and T cell lineages
Describe the pathway of multipotent HSCs to B/T cell lineage
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
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?
Growth factor mediated commitment, proliferation, initiation of antigen receptor gene rearrangement.
Selection of cells that express pre-antigen receptors.
Stages of T cell maturation (development)
Major events take place in transitional parts
What occurs during pre-lymphocyte to immature lymphocyte stage?
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
How does the thymus appear in a histology section?
What two areas can be seen
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.
The journey of T cells through development
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
Successive stages in T cell development are marked by changes in surface receptors
What are the changes in surface receptors that occur?
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
How do early T cell comitted progenitors progress from double negative?
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
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?
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.
What do the V regions of both alpha and beta chains contain?
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
TCR is similar to immunoglobulin but there are specific differences
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.
The TCR is not alone in its function
What residues bring the chains together?
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.
What are the features of antigens that are recognised by T cells?
- Most T cells recognise peptides because recognition requires close association with MHC molecules, only peptides bind to MHC molecules.
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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 - 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
- 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.
What is the anchor residue of MHC?
What is polymorphic residue?
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
What is MHC?
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
Describe the strucure of MHC
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
MHC molecules are polymorphic and polygenic
What does this mean?
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
Polymorphic residue location and MHC peptide interactions
Describe the peptide MHC interaction
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
Function and expression of MHC molecules
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
Pathway of antigen processing and presentation on top of MHC class II
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
What are the properties of the TCR?
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
What happens after gene rearrangement?
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
Germ line configuration before rearrangement
What is germ line configuration?
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.
What is the alpha delta chain encoded by?
Alpha and delta chain is encoded by chromosome 14.
- No D segments in alpha loci
- Recombination between V, J and later C
What is TCR gamma chain encoded by?
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
What mediates TCR gene segment arrangement?
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
Describe process of beta chain rearrangement
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.
Describe rearrangement of alpha chain
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.
Each T cell receptor chain is made up from different gene segments ( TCR beta chain)
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
When is TCRa chain genes rearranged?
How many times can rearrangement be attempted?
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
What is junctional diversity?
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
Compare the diversity between Ig and TCR
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
Gene rearrangements and checkpoints occur within particular regions of the thymus
Expression of what allows us to identify stages DN1-4
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
What are the consequences of successful TCR beta chain rearrangement?
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
What happens at DN4 stage after successful signalling from PreTCR?
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
