Identification of ILCs

Question 1

Where do ILCs develop?

Answer 1

In the bone marrow.

Question 2

Give the order of ILC development.

Answer 2

CLP -> ChILP -> ILC subtypes.

Question 3

Which ILCs are similar to NK cells?

Answer 3

ILC1s.

Question 4

Does E4bp4 drive ILC production?

Answer 4

Yes - it acts upon the common lymphoid progenitor.

Question 5

What are LTi cells?

Answer 5

LTi = Lymphoid Tissue Inducing Cells

• > precursors of the cells that form the lymph nodes
• > part of the ILC family.

Question 6

Give the general roles of the cytokines produced by ILCs.

Answer 6

Roles in inflammation, wound healing and fat metabolism.

Question 7

Give 3 defining characteristics of ILCs.

Answer 7

1. No Rag-dependent antigen receptors.
2. No myeloid cell markers.
3. Have lymphoid morphology.

Question 8

Give the TF that drives ILC1 production.

Answer 8

T-bet

Question 9

Give the cytokines that stimulate production of ILC1s.

Answer 9

IL-12, IL-15, IL-18 - released by epithelial cells in response to intracellular microbes, viruses and tumours.

Question 10

Give the cytokine produced by ILC1s upon activation.

Answer 10

IFNy.

Question 11

Give the main difference between NK cells and ILC1s.

Answer 11

ILC1s are not cytotoxic.

Question 12

How are NK cells distinguished from ILC1s?

Answer 12

Eomes expression is more critical than T-bet in NK cells.

Question 13

Give roles of ILC1s.

Answer 13

Macrophage activation and production of ROS.

Question 14

Give the TF that drives ILC2 production.

Answer 14

GATA3/RORa

Question 15

Give the cytokines that stimulate production of ILC2s.

Answer 15

IL-25, IL-33 and TSLP - released by epithelial cells in response to large parasites, tissue injury and during allergic responses.

Question 16

Give cytokines produced by ILC2s.

Answer 16

IL-4, IL-5 and IL-13 (type 2)

Question 17

Which ILC population produces amphiregulin?

Answer 17

ILC2s - involved in repair.

Question 18

Give the roles of ILC2s.

Answer 18

Mucus production, M2 macrophage activation, tissue repair, vasodilation and thermoregulation.

Question 19

Give the role of ILC2s in asthma.

Answer 19

• induce polarisation of naive CD4+ T cells to produce Th2 cells.
• produce IL-5 which induces eosinophila in the lungs

Question 20

Give the TF that drives ILC3 production.

Answer 20

RORyt

Question 21

Give the cytokines that stimulate production of ILC3s.

Answer 21

IL-1b and IL-23 - released in the response to extracellular microbes, including fungi.

Question 22

Give the cytokines produced by ILC3s.

Answer 22

• IL-17 and/or IL-22.
• sometimes produce IFNy or IL-13.
• produce GM-CSF.

Question 23

Give the roles of ILC3s.

Answer 23

Stimulating phagocytosis, and production of anti-microbial peptides.

Question 24

How were ILC2s first identified?

Answer 24

As IL-13 producing nuocytes - innate response to helminth infection.

Question 25

Give evidence for the roles of nuocytes during helminth infection.

Answer 25

- IL-13-eGFP mice have higher IL-13 production when given IL-25 and IL-33 to induce a Th2 response. GFP+ cells seen in the lamina propria. - Nuocytes account for why Rag -/- mice can still give a Th2 response, without T cells.

Question 26

Give the cell surface markers of nuocytes.

Answer 26

eGFP+ T1/ST2+ IL-17BR+

Question 27

Describe the experiments done with the helminth infection model to further characterise nuocytes.

Answer 27

- IL-1rl -/- (no response to IL-33) mice could not clear helminth infection until day 14. - IL-17BR -/- (no response to IL-25) mice could not clear helminth infection until day 20. - Double KOs could not clear infection. - Infection caused increased nuocyte production in the MLN. - When these nuocytes are cultured, IL-7 and IL-33 drive their expansion. - Adoptive transfer of these expanded nuocytes into IL-17BR -/- mice restores the IL-25 driven Th2 response.

Question 28

Discuss evidence for the importance of IL-25 for nuocyte activity.

Answer 28

- IL-25 does not further increase nuocyte expansion in culture with IL-7 and IL-33. - But, addition of IL-25 to IL-17BR -/- mice that have had adoptive transfer of expanded nuocytes does further increase the goblet cell hyperplasia that occurs upon nuocyte transfer.

Question 29

What is IL-17BR?

Answer 29

A subunit of the IL-25 receptor.

Question 30

What is C-Kit a marker of?

Answer 30

Cells in the early stages of development - not normally expressed by mature lymphocytes.

Question 31

What is CD45 a marker of?

Answer 31

Haematopoietic cells.

Question 32

What cell population were known to be present in cryptopatches?

Answer 32

Lin- C-Kit+ population of lymphocytes,

Question 33

What did the presence of C-Kit indicate about the lymphocytes in the cryptopatches?

Answer 33

That this was a novel cell population, different from lymphocytes already characterised - as these cells expressed C-Kit but were not in the early stages of development.

Question 34

Describe the characterisation of ILC3s.

Answer 34

- NKp46+ CD3- cells identified in the spleen and lamina propria of mice. - Staining for NKp46 and RORyt showed coexpression of these in this cell population. - Population of NKp46+ RORyt+ cells identified in mice expressing GFP under the control of RORyt; these cells were intermediate for NK1.1 - This population are reduced in the lamina propria of germ-free mice. - NKp44+ CD56+ cells from the tonsils were shown to produce IL-22. - Stimulation of PPs from mice with IL-23 in vitro resulted in IL-22 production from NKp46+ CD3- cells.

Question 35

Give a description of the cell surface markers characterised on ILC3s.

Answer 35

RORyt (hi) NKp46+ NK1.1 (int)

Question 36

Why were the RORyt (hi) NKp46+ NK1.1 (int) cells identified not NK cells?

Answer 36

- Genetic fate mapping showed NK cells never express RORyt. - These cells did not kill MHC- YAC1 cells. - These cells did not produce IFNy.

Question 37

Should there be expression of lineage defining TFs in progenitor cells.

Answer 37

Yes - these must be expressed during the precursor stage, in order to drive the gene expression that drives cells to become a particular subtype.

Question 38

Give a description of the cells identified as a possible ILC progenitor.

Answer 38

Lin-Id2+IL7Ra+CD25-α4β7+

Question 39

What other markers are expressed by Lin-Id2+IL7Ra+CD25-α4β7+ cells?

Answer 39

C-Kit and CD244

Question 40

What is CD244?

Answer 40

an NK cell receptor for NKG2D ligands, that is probably involved in intracellular signalling.

Question 41

What is PZLF?

Answer 41

TF involved in myeloid differentiation, upregulated in ILC development. Implicated in promyelocytic leukaemia.

Question 42

What happens when Lin-Id2+IL7Ra+CD25-α4β7+ cells are adoptively transferred into nude mice?

Answer 42

Reconstitution of the helper ILC network; GATA3+ ILC2s; RORγt+ ILC3s (CCR6-); CCR6+ RORγt+ CD4+ LTis – in the lamina propria of the intestine. ILC1s (NK 1.1+NKp46+Eomes-) identified in the liver.

Question 43

What are CHILPs?

Answer 43

CHILP = Common Helper Innate Lymphoid Progenitor - ILC progenitor, does not give rise to NK cells. - Lin-Id2+IL7Ra+CD25-α4β7+

Question 44

What distinguishes NK cells from helper ILCs?

Answer 44

Helper ILCs are not cytotoxic, and express IL-7Ra - not expressed by NK cells.

Question 45

Give evidence for the role of E4bp4 in ILC development.

Answer 45

In E4bp4 KO mice; - universal loss of ILC3s - loss of LTi cells - loss of ILC precursors - increased severity of infection when infected with citrobacter rodentium (causes IBS) - loss of ILC2s in the lung

Question 46

At what stage of ILC development is E4bp4 critical?

Answer 46

For the CLP -> CHILP transition.

Question 47

Give evidence that Id2 acts downstream of E4bp4 in ILC development.

Answer 47

When BM from WT + Id2-GFP mice is injected into E4bp4 KO mice, Id2 rescues the ILC population.

Question 48

What is currently thought about the roles of E4bp4 and Id2 in ILC development?

Answer 48

That both Id2 and E4bp4 are required for ILC precursors and lineage commitment - previous CHILP identification had suggested that Id2 was the earliest lineage marker. of ILCs.