T cell development and generation of repertoire diversity**

Question 1

Origin of T cells - haematopoiesis
Describe the haematopoiesis events in T cell lymphocyte development? VD

Answer 1

1. Multipoitent HSCs give rise to distinct B and T cell lineages: a. via Lymphoid precursor pathway
2. Commitment: a. LP to Pro-T cell
3. Proliferation: a. Pro-T cell to Pre-T-cell
4. Selection
5. Differentiation into distinct functional effector subpopulations: a. Effector = Mature cell that finishes maturation - performs particular immune function

Question 2

State the key factors involved within haematopoiesis of T-cells?

Answer 2

• Key factors: Stem cell factors (c-KIT)
• Cytokines (IL-7, IL-3)
• Tissue specific signals (Notch 1 and thymic stromal cells)
• TF: GATA3

Question 3

Describe the stages of T cell maturation (development) talking about major events, anatomic site and antigen dependence?

Answer 3

VD

Question 4

Site of T cell development - Thymus
:Describe the structure of the thymus?

Answer 4

• Made up of the medulla and cortex
• Contains dense network of stromal cell and lymphocytes

Question 5

Early stages of differentiation
Describe the journey of T cells through development? VD

Answer 5

• T cell precursor rearranges it’s T cell receptor genes in the thymus
• Immature T cells that recognise self MHC receive signals for survival. Those that interact strongly with self antigen are removed from the repertoire
• Mature T cells encounter foreign antigens in the peripheral lymphoid organs and are activated
• Activated T cells proliferate and eliminate infection

Question 6

State the 4 steps in how T cells develop to the proliferation step?

Answer 6

1. Notch signals by thymic stroma
2. Induction of GATA 3 (TF)
3. Commitment to T cell lineage
4. Intense proliferation

Question 7

Describe the changes in surface receptors during the successive stages in T cell development

Answer 7

• 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 the 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 are characterized by the expression of both CD4 and CD8 and later just one or the other

Question 8

How this presents on flow cytometry? VD

Answer 8

• Flow cytometry:
• Higher up or along the axis, the more of the receptor is expressed

Question 9

Structure of TCR VD
Describe the structure of TCR and the possible types? (PART 1)

Answer 9

• Upon successful rearrangement and in the periphery (if selected) T cells express high levels of TCR
• TCR is a heterodimer consisting of two transmembrane polypeptide chains covalently linked to each other by disulphide bonds

Question 10

Structure of TCR VD
Describe the structure of TCR and the possible types? (PART 2)

Answer 10

• Each chain has one Ig-like N terminal variable domain (V) and one Ig-like constant domain (C), a hydrophobic transmembrane region and a short signaling cytoplasmic region
• The V regions of both chains contain short stretches of amino acid sequence that is highly variable between receptors. These regions form the CDs or complementary determining regions. The 3 CDRs of the alpha chain and 3 of the beta chain form the peptide-MHC binding site
• Two types: alpha-beta and gamma-delta

Question 11

State the associated signalling molecules for TC found on all T-cells?

Answer 11

-> CD3 + zeta (3)

Question 12

Describe the difference between TCR and Ig in terms of components, Number of domains, number of CDs, associated signalling molecules, Affinity for antigen? State what is similar between the two?

Answer 12

VD
- Number of CDR’s similar

Question 13

Describe how the structure of TCR forms the TC signalling complex?

Answer 13

• The C regions have cysteines residues that bring the chains together
• Charged residues in the transmembrane region bind to CD3 and the zeta chain to form the TCR signaling complex
• CD3 and zeta allow for the transduction of signals upon MHC-peptide binding
• Activates T-cell binding of peptide MHC complex

Question 14

Describe 4 features that allows for the recognition of antigenic peptides by the TCR?

Answer 14

• Most T cells recognise peptides and no other molecules
• T cells recognise linear peptides and not conformational determinants of protein antigens
• T cells recognise cell associated and not soluble antigens
• CD4+ and CD8+ T cells preferentially recognise antigens samples from extracellular and cytotoxic pools respectively

Question 15

State the 4 components that allows for the close relationship between TCR and MHC?

Answer 15

• T cell contact residue of peptide
• polymorphic residue of MHC
• Anchor residue of peptide
• Peptide itself
• MHC interacts with a T cell receptor independent of the peptide

Question 16

Structure of MHC I|
What is MHC and describe the difference between what MHC I and MHC II present?

Answer 16

• MHC is the Major Histocompatibility Complex /HLA Human Leukocyte Antigen
• 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 17

Describe the structure of MHC II?

Answer 17

• Extracellular peptide binding cleft
• Ig-like domain: Linked via disulfide bond
• Cytoplasmic tail
• MHC class II has a conserved CD4 binding site: MHC class I has a conserved CD8 binding site

Question 18

Describe the features of MHC molecules in terms of polymorphism?

Answer 18

• The MHC is highly polymorphic. There are multiple variants of each gene within the population.
• The MHC is polygenic, it contains several different MHC class I and class I genes.
• Thus every individual possesses a set of MHC molecules with different ranges of peptide binding specificities
• Thus polymorphism and polygeny lead to a high degree of polymorphism

Question 19

Describe the features of MHC-peptide interactions? (PART 1)

Answer 19

• Each MHC has one cleft that binds one peptide at the time but can bind different peptides
• Peptides that bind one MHC share structural features that promote binding: MHC can bind a group of structurally similar peptides, where some peptides have preferential binding to the MHC
• Acquire peptides while assembled inside the cell

Question 20

Describe the features of MHC-peptide interactions? (PART 2)

Answer 20

• Peptide-MHC interactions are storable with low off rate
• Very small number of MHC-peptide complexes can activate a T cell
• MHC molecules can bind and display foreign and self peptide
• MHC class II binds to longer peptides than class I

Question 21

State the difference in the type of cells that express MHC class I and MHC class II on their surface? VD

Answer 21

• MHC class I: All cells but erythrocytes (RBCs)
• MHC class II: Antigen presenting cells

Question 22

Describe the steps involved in the pathway of antigen processing and presentation on top of MHC class II?

Answer 22

Step 1: Uptake of C protein into vesicular compartment of APC
Step 2: Processing of internalized proteins in endosomal/lysosomal vesicles
Step 3: Biosynthesis + transport of MHC II to endosome
Step 4: Association of processed peptide with
MHC II in vesicles
Step 5: Expression of peptide-MHC complex on surface

Question 23

States standard properties of TCR? (5)

Answer 23

• Only one form of TCR is expressed on each T cell.
• This 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 antigen binding site.
• A TCR has only one antigen binding site.
• A TCR is never secreted: Ig is secreted

Question 24

Describe the TCR gene in germ-line configuration before rearrangement?

Answer 24

• The T-cell receptor gene segments are arranged in a similar pattern to immunoglobulin gene segments
• They are rearranged by the same enzymes; Rag 1 and Rag 2
• T-cell receptors concentrate diversity in the third hypervariable region CDR3
• Gamma: delta T-cell receptors are also generated by gene rearrangement.
• Many segments could be used to form a reagent domain of chain

Question 25

Describe the key features behind the biosynthesis of TCR?

Answer 25

• Multiple variabilities with a small number of genes
• This process is antigen-independent
• “RAG 1 and 2 genes mediate the recombination events leading to rearrangement”
• Each TCR (alpha and beta chain) is made up from different gene segments

Question 26

Describe the rearrangement of TCRalpha chain genes?

Answer 26

• They do not have D gene segments
• They are rearranged only after the TCR chain gene locus has been rearranged.
• Successive rearrangements may be attempted until a productive rearrangement has been achieved

Question 27

How is extra diversity introduced in the TCR? VD

Answer 27

• Junctional diversity
• During the joining of different gene segments, addition (or removal) of nucleotides may create new sequences at junctions.
• Mediated by TdT terminal deoxynucleotidy| transferase.

Question 28

State the steps within the early development of alpha:beta chain T cells in the thymus specifying the region?

Answer 28

• CD44, along with CD25, is used to track early T cell development in the thymus
• CD44 expression is an indicative marker for effector-memory T-cells.

Question 29

Describe the signalling checkpoint once pre-TCR has occured? VD

Answer 29

1. Signalling through the pre-TCR suppresses expression of the RAG genes.
2. So, no more rearrangement at this stage, this is allelic exclusion.
   a. Allelic exclusion is a process by which only one allele of a gene is expressed while the other allele is silenced.
3. Allelic exclusion ensures that only one TCR chain gene is expressed.
4. These events together are known as beta selection

Question 30

Once successful signalling of the pre-TCR has occured, what occurs?

Answer 30

• Successful signaling of a PreTCR:
• Halts further b chain rearrangements
• Induces expression of CD4 and CD8
• Initiates alpha chain rearrangement

Question 31

Draw the flow cytomtery graphs of TCR germ line, rearranged beta locus and pre-TCR + CD4/CD8

Answer 31

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