Paper 2: Lyn-dependent signalling, DC activation of NK cells

Question 1

what is signal transduction controlled by

Answer 1

• stimulatory and inhibitory pathways regulated by kinases and phosphatases

Question 2

inactive Lyn (3)

Answer 2

• anchored to plasma membrane
• SH3, SH2, and two kinase groups linked together
• SH2 domain is bound to own tail to clamp Lyn shut

Question 3

active Lyn (3)

Answer 3

• anchored to plasma membrane
• SH3, SH2 and two kinase groups present in more linear chain
• SH2 domain no longer bound to own tail

Question 4

regulation of Lyn (2)

Answer 4

• SH2 domain used to regulate activity and for inhibition
• when tail is bound, Lyn is inactive; when tail is’t bound, Lyn is inactive

Question 5

Lyn (3)
- protein type
- expression in cells
- activation

Answer 5

• Src-family tyrosine kinase (SFK)
• expressed in all leukocytes except T cells
• activated by ligand binding to adhesion molecules, cytokine receptors, immunoreceptors, and TLRs

Question 6

does Lyn amplify or restrict signal transduction

Answer 6

• depending on cell microenvironment, developmental stage, and type of stimulus, Lyn can either restrict or amplify signal transduction

Question 7

what activates NK cells to their full potential

Answer 7

• secretion of cytokines from CD11c^high DCs activated by TLRs is required to induce NK cell IFN production and cytotoxicity

Question 8

when does negative regulation by Lyn occur (2)

Answer 8

• when Lyn phosphorylates tyrosine residues within ITIMs present in inhibitory proteins at the plasma membrane
• results in antagonization of signalling

Question 9

when does positive regulation by Lyn occur (2)

Answer 9

• when Lyn phosphorylates ITAMs on membrane proteins
• results in amplifying signals

Question 10

Lyn is important for establishing inhibitory signalling, what occurs in Lyn-/- mice (4)

Answer 10

• enhanced BCR signalling and failure to establish inhibitory signalling
• leads to autoimmunity and over-activity of BCR; BCRs with antibodies to self are allowed to develop
• develop severe fatal autoimmune lupus and nephritis
• spleen and blood are packed with leukocytes that are normally contained and controlled by infection

Question 11

what is the general phenotype of loss of Lyn

Answer 11

• perturbation in signalling balance and severe pathology as myloid cells proliferate uncontrollably

Question 12

what does injection of LPS into mice model

Answer 12

• mimics pathophysiological consequences of sepsis

Question 13

what is the purpose of figure 1

Answer 13

• to investigate if Lyn regulates endotoxin sensitivity in vivo

Question 14

what was observed in figure 1 (2)

Answer 14

• Lyn^up/up mice were ~80-fold more sensitive to LPS than Lyn+/+ or Lyn-/- mice shown by the amounts of LPS required to drop body temperature
• Lyn^up mice had increased levels of macrophages, neutrophils, lymphocytes, eosinophils and TNF-a compared to wt mice after LPS inhalation

Question 15

what were the findings from figure 1

Answer 15

• Lyn^up/up mice exhibit enhanced endotoxin-induced inflammation and morbidity

Question 16

what was the purpose of figure 2

Answer 16

• to investigate if Lyn regulated endotoxin-induced cytokine production

Question 17

what methods were used for figure 2A, 2B, and 2C (3)

Answer 17

• ELISA
• western blot
• flow cytometry

Question 18

what was observed in figure 2A

Answer 18

• Lyn^up/up serum contained significantly higher concentrations of pro-inflammatory cytokine IL-12, IFN-g, IL-1a, IL-1B, TNF-a, IFN-a, and IL-6 and lower concentrations of IL-10 compared to Lyn+/+ mice after LPS injection

Question 19

what was observed in figure 2B

Answer 19

• LPS-induced expression of proinflammatory mediator iNOS was elevated in kidney, spleen, lung, and liver of Lyn^up/up mice after LPS injection compared to wt

Question 20

what was observed in figure 2C

Answer 20

• myloid cells in Lyn^up/up BM had enhanced expression of MHC class 1 and Mac-1 after LPS injection

Question 21

what are the findings from figure 2

Answer 21

• Lyn promotes production of pro-inflammatory cytokines in response to endotoxin

Question 22

why was IFN-g important in this paper (2)

Answer 22

• IFN-g production in response to LPS is directly linked to magnitude of innate immune responses in vivo
• blocking of IFN-g with neutralizing Abs can prevent fatal LPS-induced sepsis in mice

Question 23

identifying intracellular proteins (4)

Answer 23

• to capture steady state of the cell, cells must be fixed/preserved or have Golgi Apparatus preserved
• cells can then be stained be permeabilized
• stain with fluorescent antibodies
• analysis on flow cytometer

Question 24

what is the purpose of figure 3

Answer 24

• to investigate how Lyn modulated IFN-g production in LPS-treated mice

Question 25

according to the literature, what cells are known to produce IFN-g (2)

Answer 25

• NK cells
• T cells

Question 26

what was observed in figure 3A (3)

Answer 26

• Lyn^up/up CD4+ and CD8+ T cells showed little LPS-induced IFN-g production
• 85% of of splenic NK cells in Lyn^up/up mice expressed IFN-g compared to 20% in Lyn+/+ mice after LPS
• Lyn^up/up mice expressed more IFN-g per cell, as indicated by MFI

Question 27

what was observed in figure 3B and 3C

Answer 27

• Rag-/- Lyn^up/up mice, which have no B, T, or NKT cells, still experienced LPS-induced morbidity and LPS-induced NK cell IFN-g production

Question 28

what were the findings from figure 3

Answer 28

• NK cells are the primary source of IFN-g in LPS-challenged Lyn^up/up mice

Question 29

how do NK cells acquire effector functions such as IFN-g production

Answer 29

• must be stimulated by either DCs or macrophages

Question 30

what was the purpose of figure 4A and 4B

Answer 30

• investigate whether macrophages or DC subsets were responsible for stimulating NK cells to produce IFN-g in LPS-stimulated Lyn^up/up mice

Question 30

what was observed in figure 4A

Answer 30

• different DC subsets were effective at inducing NK cells to secrete IFN-g in response to LPS, but macrophages were not

Question 31

what method was used for figure 4A and 4B

Answer 31

• ELISA

Question 32

what was observed in figure 4B

Answer 32

• of the DC subsets, CD11c^high DCs were the most potent activators of NK cell IFN-g production after LPS treatment

Question 33

is it possible to remove entire populations of cells from mice (2)

Answer 33

• yes, use cell-specific DTR model
• have mice cell of interest express DTR so that when DT is injected, the cells undergo apoptosis

Question 34

what was the purpose of figure 4C and 4D

Answer 34

• to investigate if DCs are required for excessive NK cell IFN-g production in Lyn^up/up mice

Question 35

what was observed in figure 4C and 4D (2)

Answer 35

• without DC ablation, Lyn^up/up contained significantly more IFN-g+ NK cells and elevated IFN-g in serum than Lyn+/+ mice after LPS treatment
• both Lyn^up/up and Lyn+/+ mice had reduction in IFN-g+ NK cells and IFN-g serum levels when DCs were ablated after LPS treatment

Question 36

what were the findings in figure 4C and 4D

Answer 36

• DC lineage is required for enhanced NK cell IFN-g production in Lyn^up/up mice

Question 37

what was the purpose of figure 4E and 4F

Answer 37

• investigate whether DCs and NK cells are also required for LPS-induced morbidity in Lyn^up/up mice

Question 38

what was observed in figure 4E and 4F

Answer 38

• 75% of Lyn^up/up mice rescued from LPS-induced morbidity after DC depletion
  -100% of of Lyn^up/up mice rescued from LPS-induced morbidity after NK cell depletion

Question 39

what were the findings from figure 4E and 4F

Answer 39

• DC and NK cells are required for LPS-induced morbidity in Lyn^up/up mice