Flashcards in Identification of ILCs Deck (48):
Where do ILCs develop?
In the bone marrow.
Give the order of ILC development.
CLP -> ChILP -> ILC subtypes.
Which ILCs are similar to NK cells?
Does E4bp4 drive ILC production?
Yes - it acts upon the common lymphoid progenitor.
What are LTi cells?
LTi = Lymphoid Tissue Inducing Cells
-> precursors of the cells that form the lymph nodes
-> part of the ILC family.
Give the general roles of the cytokines produced by ILCs.
Roles in inflammation, wound healing and fat metabolism.
Give 3 defining characteristics of ILCs.
1. No Rag-dependent antigen receptors.
2. No myeloid cell markers.
3. Have lymphoid morphology.
Give the TF that drives ILC1 production.
Give the cytokines that stimulate production of ILC1s.
IL-12, IL-15, IL-18 - released by epithelial cells in response to intracellular microbes, viruses and tumours.
Give the cytokine produced by ILC1s upon activation.
Give the main difference between NK cells and ILC1s.
ILC1s are not cytotoxic.
How are NK cells distinguished from ILC1s?
Eomes expression is more critical than T-bet in NK cells.
Give roles of ILC1s.
Macrophage activation and production of ROS.
Give the TF that drives ILC2 production.
Give the cytokines that stimulate production of ILC2s.
IL-25, IL-33 and TSLP - released by epithelial cells in response to large parasites, tissue injury and during allergic responses.
Give cytokines produced by ILC2s.
IL-4, IL-5 and IL-13 (type 2)
Which ILC population produces amphiregulin?
ILC2s - involved in repair.
Give the roles of ILC2s.
Mucus production, M2 macrophage activation, tissue repair, vasodilation and thermoregulation.
Give the role of ILC2s in asthma.
- induce polarisation of naive CD4+ T cells to produce Th2 cells.
- produce IL-5 which induces eosinophila in the lungs
Give the TF that drives ILC3 production.
Give the cytokines that stimulate production of ILC3s.
IL-1b and IL-23 - released in the response to extracellular microbes, including fungi.
Give the cytokines produced by ILC3s.
- IL-17 and/or IL-22.
- sometimes produce IFNy or IL-13.
- produce GM-CSF.
Give the roles of ILC3s.
Stimulating phagocytosis, and production of anti-microbial peptides.
How were ILC2s first identified?
As IL-13 producing nuocytes - innate response to helminth infection.
Give evidence for the roles of nuocytes during helminth infection.
- 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.
Give the cell surface markers of nuocytes.
eGFP+ T1/ST2+ IL-17BR+
Describe the experiments done with the helminth infection model to further characterise nuocytes.
- 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.
Discuss evidence for the importance of IL-25 for nuocyte activity.
- 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.
What is IL-17BR?
A subunit of the IL-25 receptor.
What is C-Kit a marker of?
Cells in the early stages of development - not normally expressed by mature lymphocytes.
What is CD45 a marker of?
What cell population were known to be present in cryptopatches?
Lin- C-Kit+ population of lymphocytes,
What did the presence of C-Kit indicate about the lymphocytes in the cryptopatches?
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.
Describe the characterisation of ILC3s.
- 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.
Give a description of the cell surface markers characterised on ILC3s.
RORyt (hi) NKp46+ NK1.1 (int)
Why were the RORyt (hi) NKp46+ NK1.1 (int) cells identified not NK cells?
- Genetic fate mapping showed NK cells never express RORyt.
- These cells did not kill MHC- YAC1 cells.
- These cells did not produce IFNy.
Should there be expression of lineage defining TFs in progenitor cells.
Yes - these must be expressed during the precursor stage, in order to drive the gene expression that drives cells to become a particular subtype.
Give a description of the cells identified as a possible ILC progenitor.
What other markers are expressed by Lin-Id2+IL7Ra+CD25-α4β7+ cells?
C-Kit and CD244
What is CD244?
an NK cell receptor for NKG2D ligands, that is probably involved in intracellular signalling.
What is PZLF?
TF involved in myeloid differentiation, upregulated in ILC development. Implicated in promyelocytic leukaemia.
What happens when Lin-Id2+IL7Ra+CD25-α4β7+ cells are adoptively transferred into nude mice?
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.
What are CHILPs?
CHILP = Common Helper Innate Lymphoid Progenitor
- ILC progenitor, does not give rise to NK cells.
What distinguishes NK cells from helper ILCs?
Helper ILCs are not cytotoxic, and express IL-7Ra - not expressed by NK cells.
Give evidence for the role of E4bp4 in ILC development.
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
At what stage of ILC development is E4bp4 critical?
For the CLP -> CHILP transition.
Give evidence that Id2 acts downstream of E4bp4 in ILC development.
When BM from WT + Id2-GFP mice is injected into E4bp4 KO mice, Id2 rescues the ILC population.