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Flashcards in NK Cell Development Deck (30):
1

Give markers of the common lymphoid progenitor.

IL7R, CD27, C-Kit, Flt3.

2

What is Flt3?

Cytokine receptor for the Flt3 ligand - important for hematopoietic development.

3

What is CD27?

TNF family receptor and T cell co-stimulator.

4

What is NK1.1?

C-type lectin activatory receptor.

5

What is the therapeutic relevance of understanding the transcriptional control of lymphoid development?

May want to be able to drive differentiation to give a specific cell type and immune response.

6

Why can each TF recognise multiple genes?

TFs only recognise 6-8bp of DNA - this is not that specific, and will occur in multiple genes.

7

Where does further control come from during differentiation?

Interactions with other transcription factors and epigenetic changes.

8

Describe E4bp4.

TF with bZIP domain, where a leucine zipper allows DNA binding. Member of the PAR family.

9

What is E2A-HLF?

A fusion TF that forms during a rare type of leukaemia - recognises same DNA consensus sequence as E4bp4.

10

How is E4bp4 involved in circadian rhythm regulation?

Controls expression of the period genes.

11

Give cells types that express E4bp4.

NKT cells in the spleen and NK cells in the spleen and BM - suggests E4bp4 is involved in NK cell development.

12

Give evidence for the importance of E4bp4 in NK cell development.

- E4bp4 KO mice lose NK cells in spleen and BM.
- E4bp4 KO mice cannot reject RMA/s tumour cells (no
MHC).
- E4bp4 KO mice only have NKP populations in the BM, and cannot produce iNK or mNK cells.
-> concluded that E4bp4 acts between progenitors and immature NK cells.

13

How do E4bp4 expression levels change throughout NK cell development?

Increased expression in immature and mature NK cells. Lower expression seen in NK progenitors.

14

Give evidence that E4bp4 knockout is intrinsic and does not affect the stem cell niche.

- Normal developmental processes occur when WT BM is transplanted into irradiated E4bp4 KO mouse.
- Ly5.1+ irradiated recipient mice survive when given BM from E4bp4 KO mice - BM niche is functional and unaffected by E4bp4 KO.

15

How are NK cells generated in culture?

Using IL-7 and IL-15 to drive progenitors to differentiate into NK cells, using stromal feeder cells as an artificial niche.

16

Can transduction of E4bp4 rescue the KO phenotype? Give evidence.

Yes - transduction of E4bp4 into E4bp4 KO cells gives rescue of NK cell development, even in the absence of IL-15.

17

How can it be tested whether or not E4bp4 affects survival?

Transduction of anti-apoptotic proteins into E4bp4 KO progenitor cells - doesn't rescue NK production.

18

How can it be tested whether or not E4bp4 affects proliferation?

Using BrdU staining to measure the effect of E4bp4 overexpression on proliferation rate - no effect was seen.

19

Why was it tested if E4bp4 affected survival or proliferation?

To determine whether E4bp4 drives NK production purely through transcriptional control.

20

Why was it important to test the effects of E4bp4 KO on CD8+ T cell activity?

IL-15 is important for CD8+ T cell activity, as well as for NK cell development. Needed to check if E4bp4 knockout was affecting T cell cytotoxicity through modulation of IL-15 production.

21

How was CD8+ T cell activity tested in E4bp4 KO mice?

Mice had spleenocytes removed, and pulsed with an antigenic peptide from influenza virus. Upon infection of influenza virus, spleenocytes were injected, and spleenocytes that were previously pulsed with antigenic peptide were eliminated in both WT and E4bp4 KO mice - loss of E4bp4 did not affect the CD8+ cytotoxic response.

22

How was the NKP population originally defined?

BM Lin-CD122+

23

Give the evidence that suggested NKPs were not true NK cell progenitors.

When isolated in culture, only 1 in 12 of these progenitor cells gave rise to NK cells, and the ‘NKPs’ could differentiate to give other cell types.

24

How were pre-NKPs identified?

Isolation of the BM Lin-CD122+ cells from mice - gated for the cells negative for CD19, CD3, etc. to remove B cells, T cells, macrophages - then gate for CD27+ IL7Ra+.
Gating for CD122 against Flk2 (-) :
- Gives two separate populations of NKPs; the pre-NKPs (low CD122) and the NKPs (high CD122).
- Using these NKPs, gives 1 in 1.5 cells differentiating to give NK cells.

25

Are these NKPs (CD122 high) present in E4bp4 KO mice?

Not present in E4bp4 KO mice

26

How was it shown that CD122 high NKPs do not require IL-15?

Present in IL-15R knockout mice, which have no NK cells.

27

How was it shown that T-bet acts downstream of E4bp4?

CD122 high NKPs are present in T-bet knockout mice, which have no NK cells.

28

What transcription factors is E4bp4 expressed before?

Id2, Eomes and T-bet.

29

How were the TFs downstream of E4bp4 identified?

By genetic complementation of E4bp4 KO NKPs cultured on stromal cells with IL-15. If a TF is downstream, its addition will rescue the knockout phenotype.

30

Which TFs are directly expressed by E4bp4?

Id2 and Eomes.