L4 - Lymphocyte development

Question 1

Where do all cells come from?

Answer 1

All cells come from bone marrow & become pluripotent haemopoietic stem cells

Question 2

B cell development

Answer 2

Develop from haematopoietic stem cells in bone marrow that express PAX5 transcription factor

Continuous process: 3 x 1010 produced per day

Lots of cells don’t leave the bone marrow – most don’t survive when screened for receptors & self-reactive cells

Involves rearrangement & expression of Ig genes

Question 3

Why do lots of cells not leave the bone marrow?

Answer 3

Most don’t survive when screened for receptors & self-reactive cells

Question 4

What is CD45?

Answer 4

A key marker of lymphocytes

Question 5

What is CD19?

Answer 5

A slightly later marker of B cells – not expressed in early B cells

Question 6

Where do the final stages of development from immature B cells happen?

Answer 6

In peripheral lymphoid tissues (eg. spleen)

Question 7

What does PAX5 do?

Answer 7

PAX5 turns on genes that are going to turn into a B cell – interacts with bone marrow stromal cells

Question 8

What happens once PAX5 has turned on the right genes?

Answer 8

During the process the B cell will now start to make the randomly re-arranged cell receptor to put on its surface

First ones it makes are IgM & IgG isotypes

Question 9

What happens if the receptors produced can recognise antigens in the bone marrow?

Answer 9

If receptor can recognise antigens, those B cells are given a signal to die

If they recognise nothing they exit the bone marrow & survive – has the ability to recognise something that isn’t expressed in that environment

THIS IS NEGATIVE SELECTION

Question 10

Where do the B cells that survived negative selection go?

Answer 10

Go through the circulatory system to lymphoid organs/nodes

Question 11

What happens if the receptors produced can recognise antigens in lymphoid organs/nodes?

Answer 11

If the receptor now recognises something, it is a potential antigen it needs to respond to

Cells with the right receptor proliferate – clonal expansion

Differentiate & become plasma cells to secrete the antibody instead of displaying it on the surface

THIS IS POSITIVE SELECTION

Question 12

Why do not all B cells become plasma cells secreting antibody?

Answer 12

Some become memory cells & keep BCR on their surface – can remain for 10-20 years

Majority of cells won’t recognise an antigen as you’re not infected so will die after a few days

B cells die at the same rate they are made

Question 13

Pre-B cells

Answer 13

H chain genes rearrange first

Ig-mu chain expressed with surrogate light chain – product of VpreB & lamda-5 genes

Question 14

Why is a surrogate light chain used?

Answer 14

Tests whether heavy chain is going to be any good

Simple models to test binding of the heavy chain

If it fails it dies straight away

Question 15

Where does the H chain bind the surrogate light chain?

Answer 15

H chain has to move to the surface to bind to the surrogate light chains

Associates with Ig-alpha & Ig-beta (invariant molecules) to do this

Question 16

What happens if H chain binds the surrogate light chain?

Answer 16

Sends a signal to the B cell if it can bind to the surrogate light chain & can move to the next stage of development

Next: make proper light chains instead of surrogate light chains

Question 17

Pre-B cell receptor pre-BCR

Answer 17

Signal from pre-BCR:
• Turns off RAG1 & RAG2 genes – don’t make another heavy chain
• 5-6 rounds of cell division
• Surrogate light chain expression stops
• RAG1 & RAG2 turned on again – allows light chains to be made
• L chain rearrangement starts

Question 18

What are RAG genes needed for?

Answer 18

Gene rearrangement - are regulated in time

Question 19

What types of L chain can B cells express?

Answer 19

Kappa or lamda

Each rearrangement has a 1 in 3 chance of being successful

As kappa chain rearranges first, more B cells express kappa than lambda

Question 20

Immunoglobulin gene rearrangements

Answer 20

Error prone

Dies if it doesn’t productively re-arrange

Pre-B cells with non-productive rearrangements of light chain genes can be ‘rescued’ by up to 5 further rearrangements at the same locus – if after all these attempts still out of frame then lambda locus begins to rearrange

Question 21

What happens to immature B cells that bind multivariant self-antigens?

Answer 21

Undergo either:
• Clonal deletion – cell dies by apoptosis
• Receptor editing – further L chain rearrangements of variable regions (another chance)

Question 22

What happens to immature B cells that bind soluble self antigens?

Answer 22

Become unresponsive - anergic

Can escape death – can downregulate BCRs that are potentially autoreactive

Question 23

T cell development

Answer 23

Initially similar to that of B cells 
– Originate from bone marrow stem cells 
– Re-arrange receptor genes 
– Express pre-T receptor 
– Elimination of self-reactive T cells by negative selection

Question 24

How is T cell development different from B cell development?

Answer 24

Have 2 more robust selection methods – positive & negative selection

Undergo development/selection in the thymus
• Either rearrange alpha/beta TCR genes (CD4+ & CD8+) or gamma/delta TCR genes

T cells expressing alpha/beta TCR must bind with self MHC expressed in thymus
• Positive selection

Question 25

TCR binds self-MHC

Answer 25

Cannot bind with too much affinity otherwise will die Vast majority of T cells fail this test – 95% die 5% leave & go into blood & lymph nodes

Question 26

Alpha/beta T cell development

Answer 26

Precursors produced in bone marrow migrate to thymus Once in the thymus they develop into thymocytes: 1. Re-arrange TCR genes (beta first) & express TCR 2. Acquire other markers – eg. CD3, CD4 & CD8 3. Undergo positive & negative selection

Question 27

What is the thymus?

Answer 27

Bi-lobed organ in anterior mediastinum Each lobe divides into many lobules Each lobule has outer cortex & inner medulla

Question 28

What cells are present in the thymus?

Answer 28

Lymphoid cells Epithelial cells Macrophages & dendritic cells

Question 29

What happens in the thymus?

Answer 29

Where T cells are selected for their ability to recognise self-MHC & then if they survive they are killed if they recognise it too well

Question 30

T cell maturation in thymus

Answer 30

Pro-thymocytes enter cortex via blood vessles from bone marrow Re-arrange TCR genes – Firstly re-arrange TCR-beta genes – Expressed along with the pre-TCR-alpha (invariant): pre-T cell receptor – Cells proliferate & then re-arrange TCR-alpha genes Express TCR together with CD3 Also express CD4 & CD8

Question 31

What does TCR expression require?

Answer 31

CD3 complex – essential for transmitting signals – Delta, epsilon & gamma chains – Zeta chain dimer CD3 transmits signal to T cell nucleus following TCR recognition of p/MHC

Question 32

Gamma/delta TCR

Answer 32

* Similar structure to alpha/beta receptor * No CD4 or CD8 markers * Less diversity than alpha/beta * Expressed on separate T cell population * Recognise different antigens * Only go through positive selection Lineage commitment to gamma/delta or alpha/beta depends upon which genes are first to rearrange successfully

Question 33

T cells expressing a randomly rearranged alpha/beta TCRs may be able to recognise what MHC?

Answer 33

1. Recognise self MHC plus peptide from ‘foreign’ Ag – immunity 2. Recognise self MHC plus peptide from ‘self’ Ag – autoimmunity 3. Not be able to recognise self-MHC – useless Need to keep T cells with TCR 1, eliminate those with TCR 2 & 3 – positive & negative selection Reality is a fine balance based in TCR/MHC affinity

Question 34

Positive selection of T cells

Answer 34

Positive selection of cells which recognise self MHC (+ self-peptide) Occurs when double-positive (DP, CD4+, CD8+) T cells recognise MHC on cortical epithelial cells in thymus – apoptosis if not recognised Positively selected cells move to medulla

Question 35

Proposed models for the role of CD4 & CD8 in thymic positive selection of DP thymocytes leading to SP T cells

Answer 35

2 possible theories: 1. Instructive model Interaction of 1 co-receptor, with MHC molecules on thymic stromal cells during MHC I or II mediated positive selection results in the down regulation of the other co-receptor 2. Stochastic (random) model Down-regulation of the co-receptor occurs randomly in DP cells, prior to MHC-mediated positive selection Those cells that have down-regulated CD4 will be selected on MHC I, & those that have down-regulated CD8 will be selected on MHC II

Question 36

Negative selection of T cells

Answer 36

Negative selection of T cells which recognise self MHC (+ self-peptide) on thymic dendritic cells/macrophages with high affinity (as these T cells may induce autoimmunity if not removed) TCR binding to MHC/self-peptide with high affinity causes T cell to die by apoptosis (clonal deletion)

Question 37

TCR affinity for p/MHC is basis for selection

Answer 37

All T cells recognising self-MHC/self-peptide are positively selected Those with the highest affinity TCR are then negatively selected Ultimate goal = a population of T cells with low affinity for self-peptide + self MHC These cells represent the safest/most useful to keep with the highest probability they will have a high affinity for self MHC when presenting peptides derived from pathogens

Question 38

What happens to T cells that survive thymic selection?

Answer 38

< 5% Express TCR capable of binding self MHC Are depleted of self-reactive cells Exit thymus as mature, single positive (SP) T cells

Question 39

What are the types of SP T cells?

Answer 39

CD4+ SP T cells recognise Ag in association with MHC class II CD8+ SP T cells recognise Ag in association with MHC class I