3. Peripheral CD4+ T cell differentiation Flashcards

1
Q

How quickly can T cells expand?

A

They can go from 200 naive cells to 10,000 effector cells in 4 days.

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2
Q

Overview of clonal T cell expansion

A
  1. Naive T cell expressing a single TCR on the surface
  2. Interaction with an APC expressing an MHC.
  3. If the TCR and MHC interact in a stimulatory way it will activate the T cell.
  4. Naive T cell receives a signal to expand and differentiate
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3
Q

Why is differentiation of CD4 T cells also important?

A
  1. It tailors effector mechanisms to pathogens.
  2. Limiting collateral damage and keeping the response just enough.
  3. It supports immunological memory and makes vaccination possible
  4. Together, these all optimise the adaptive immune response.
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4
Q

How were the different types of CD4 cells originally characterised?

A

A 1986 publication at the point it became possible to clone enough CD4 cells in culture to identify the proteins in them.
The CD4 T cells were classified by what they produce.
Showed that different cytokines are made depending on differentiation and function.

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5
Q

Have all the CD4+ subtypes been discovered?

A

No, more are still being found

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6
Q

Why is correct differentiation of CD4 T cells important?

A
  1. The type of CD4 response can determine the outcome of infection.
  2. Th1 responses can clear and control certain infections, whereas Th2 responses won’t.
  3. This changes the infection outcomes eg death.
  4. Due to genetics not everyone has the same ability to make the different responses.
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7
Q

What are naive CD4 T cells?

A

Multipotent

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8
Q

Does the peptide presented to the T cell determine its differentiation fate?

A

No. At this point the T cell is still multipotent and what activates the T cell doesn’t determine its fate.
However, the affinity at which this activation occurs does have some bearing on the fate of the cell.

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9
Q

Does the environment the T cell is in determine its differentiation fate?

A

Yes, it plays a role through the cytokines in the environment but there are also other factors.

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10
Q

What factors can determine a CD4 T cells fate?

A
  1. APC phenotype - how the antigen is presented
  2. Concentration of antigen
  3. TCR activity
  4. Genetics
  5. Cytokine signalling
  6. Adjuvant immunity, e.g. TLR signalling and innate immunity
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11
Q

What 3 signals are needed for T cell activation?

A
  1. TCR- MHC recognition
  2. Costimulation
  3. Instruction through cytokines
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12
Q

Where does CD4 T cell differentiation occur?

A

In the lymph nodes but in different areas for different cell types

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13
Q

Th1 differentiation in the lymph node

A
  1. Dendritic cell 1 generates peripheral cells deep in the T cell zone.
  2. This produces effector Th1 cells and T follicular helper cells.
  3. The Tfh aid specific antibody production.
  4. This is the type 1 response and mostly for intracellular pathogens.
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14
Q

Th2 differentiation in the lymph nodes

A
  1. Dendritic cell 2 generates Th2 effector cells adjacent to the B cell follicles.
  2. Also, some generation of Tfh cells.
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15
Q

What initiates Tfh differentiation?

A

Lymph node resident cDC2

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16
Q

Differentiation signals: Th1

A

IL12

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17
Q

Differentiation signals: Th2

A

IL4

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18
Q

Differentiation signals: Th17

A

IL6, TGFß and IL23

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19
Q

Differentiation signals: Tfh

A

IL21

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20
Q

Differentiation signals: Treg

A

IL21

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21
Q

Effector Molecules: Th1

A

IFNy

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22
Q

Effector Molecules: Th2

A

IL4, IL5, IL13

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23
Q

Effector Molecules: Th17

A

IL17,IL22, GM-CSF

24
Q

Effector Molecules: Tfh

A

IL21, IL10

25
Q

Effector Molecules: Treg

A

TGFß

26
Q

How can we identify different CD4 T cell subsets?

A
  1. Different effector molecules released
  2. Different transcription factors produced
  3. Different gene expression profiles
27
Q

What do the CD4 T cell activation signals cause?

A

Modification of the expression of certain genes that leads to the production of effector cytokines depending on the differentiation signals.

28
Q

How do differentiated T cells respond differently to activation signals?

A
  1. They are more sensitive to stimuli.
  2. Larger amounts of cytokines are produced in response to the signals.
  3. Other cytokine production if switched off.
  4. This forms part of the memory response.
29
Q

How do differentiated T cells form part of immune memory?

A
  1. They are more sensitive to environmental stimuli.
  2. They respond to reinfection more rapidly.
30
Q

How do differentiated T cells cause problems?

A

They make autoimmune diseases harder to treat as the disease progresses because they are much easier to stimulate.

31
Q

How do daughter cells know what phenotype their mother was?

A

Epigenetic remodels the DNA to favour the expression of particular effector functions.

32
Q

Epigenetic changes during differentiation: silencing

A
  1. A naive CD4 T cell starting to go through Th1 differentiation still can produce low levels of IL-4.
  2. Th1 cells don’t express IL-4.
  3. Once the differentiation is complete the IL-4. locus is silenced by hypermethylation especially in the non-coding sequence 2 (CNS2).
33
Q

Epigenetic changes during differentiation: Expression

A
  1. A naive CD4 T cell starting to go through Th2 differentiation produces low levels of IL-4.
  2. Extensive demethylation drives an increase in IL-4 transcription.
34
Q

Studying Epigenetic Mechanisms: Visualisation

A

Repositioning with respect to loose or condense chromatin.

35
Q

Studying Epigenetic Mechanisms: DNAse hypersensitivity

A

DNA accessibility changed by modification in methylation state.

36
Q

Studying Epigenetic Mechanisms: ChIP and ChIP-seq assays

A

Identifying histone tail modifications like acetylation and methylation.

37
Q

Studying Epigenetic Mechanisms: ATAC-seq

A

assess genome-wide chromatin openness. The data shows areas of open chromatin available for modification. The different reads show differences in phenotype.

38
Q

Why do adaptive immune cells need to expand?

A
  1. It cannot sustain the large amount of cells needed for an effective response all the time.
  2. Most effector cells are short lived and die in a few days.
39
Q

Studying T cell differentiation and genetic changes: fate mapping

A
  1. This is used to track genetic changes throughout a cell’s life.
  2. You take the promoter from the gene you want to trace the expression of eg IL-17.
  3. Put the IL-17 promoter on the detectable marker gene like Cre
  4. Cre Cuts a Fluorescent protein (eYFP) gene to activate it. This makes the activation of IL-17 detectable.
  5. Every cell that is expressing IL-17 promoter will turn green so you can see which cells are Th 17 cells.
40
Q

What is Cre?

A

A recombinase enzyme that removes DNA from between 2 loxP sites.

41
Q

How can you extent fate mapping?

A
  1. To tract further differentiation through gene expression.
  2. Use it to understand the history of the cell.
  3. Use a 2nd colour to trace 2 genes together.
42
Q

What is a problem with fate mapping?

A

It doesn’t show the cells that die.

43
Q

What is eYFP?

A

extended Yellow Fluorescent Protein

44
Q

Studying T cell differentiation and genetic changes: Gene expression profiling

A
  1. It looks at all the genes being expressed in a cell at a certain point in time.
  2. Only possible due to high throughput sequencing.
  3. Sequence the mRNA to measure gene expression.
45
Q

What are the benefits of gene expression profiling?

A
  1. You can compare cells to each other to see which genes could have different pathogenicity or effect phenotype.
  2. You can see multiple genes at a time and see their combined effect.
  3. You can see how the environment can alter gene expression.
46
Q

What are the disadvantages of gene expression profiling?

A
  1. Sometimes there is correlation but not causation which can be hard to distinguish.
  2. There is lots of data with few samples so it needs to be summarised.
47
Q

How can you summarise gene expression profiling?

A

A principle component analysis plot which looks at which genes are associated with the signature.

48
Q

What methods are used for gene expression profiling?

A
  1. scRNA-seq
  2. Hi-dimensional flow cytometry and cytoseek
  3. scTransfer and counting
49
Q

What is scRNA-seq?

A

Single-cell RNA-seq. It takes a cell and measures the expression. You can do this on many cells to build a picture of gene expression how it changes.

50
Q

scRNA-seq 2d projections

A
  1. Measure the expression of every cell for a variety of genes.
  2. Ask a computer to determine which other cell it is most similar to.
  3. This generates clusters of cells with similar gene expression.
  4. Take this picture and look for a particular gene and find where in these clusters and therefore which cell types it is expressed.
  5. Can take cells at different stages in differentiation to see how it changes gene expression.
51
Q

What is an example of differentiated CD4 T cells being needed to clear an infection?

A

A macrophage infected with mycobacterium can resist the action of some T cells but Th1 cells produce IFNy that stimulates the macrophage to destroy TB.

52
Q

What is an IL-17 blockade associated with candidasis?

A
  1. The expression of peptides in the thymus changes causing a change in T cell repertoire.
  2. This causes active CD4 T cell responses to self-antigens.
  3. Antibodies to IL-17 are produced causing an IL-17 deficiency.
  4. This causes a lack of Th17 function.
  5. Th17 is important in the clearance of fungal infections like candidiasis.
53
Q

What do strong phenotypic effects of CD4 T cells cause?

A
  1. Organ specific autoimmune disease.
  2. Asthma and airway hyper-reactivity
  3. Chronic diseases
54
Q

Can CD4 T cell differentiation be used in therapeutics?

A
  1. Experimentally they can be effective but an effective therapy is more difficult to develop.
55
Q

What are some successful therapies?

A
  1. Anti-TNF therapies are effective but are less effective when the disease is more severe.
  2. Anti-IL-17 to treat psoriasis.
  3. These therapies are very specific so giving them to the wrong patient can cause problems.