Exercise 1 & 2 - DCs & BM Cell isolation

Question 1

What is a dendritic cell (DC)?

Answer 1

A phagocytotic cell, present in peripheral tissue and in secondary lymphoid organs, with high T-cell stimulating capacity.

A professional antigen-presenting cells (APC) - presenting antigen at MHC II.

Question 2

Are DCs good at degrading pathogens?

Answer 2

• No, DCs are the best APCs! But their phagocytotic abilities are not as good as neutrophils or macrophages.

Question 3

How is an antigen presented?

Answer 3

Endocytosis or phagocytosis –> endosome/phagosome will fuse with lysozome –> degradation –> presentation on MHC II –> transport to surface.

Question 4

How are DCs activated?

Answer 4

Immature DCs in peripheral tissues genkender antigen via PRR (Pattern Recognition Receptor) der genkender PAMPs –> Meets antigen –> Upregulates CCR8 (Chemokine Receptor) –> Migrates by lymph to nearby lymph node –> Activated DC will encounter naive T cells, some might bind the MHC II bound antigen and be activated, proliferate and differentiate.

Remember: T cells need both TCR-MHC II:Antigen contact + costimulation + cytokines

Question 5

How is the activating signal transduction on DC?

Answer 5

• Often through TLR (Toll like receptors) on the surface or on the endosome.

Question 6

How is the built of TLR?

Answer 6

• They are always dimers, either homo- or hetero dimers. They need a dimer to send a signal in the cell.

Question 7

How are DCs matured by danger signals?

Answer 7

• Inflammatory activation stimuli –> Early activation results in better endocytotic and phagocytotic activities + cytokine production (attracts innate immunesystem) –> intermediate activated DCs (after 8ish hours) loose endocytotic functions and migrate to lymph nodes –> fully mature DCs in lymph nodes will increase in antigen presentation and processing, activate T cells and after 1-2 days after stimulation, the DC will become apoptotic and die.

Question 8

How many DC’s are in the peripheal tissue?

Answer 8

• Not that many, they are present in relatively low number, but can cover quite an area with it’s protrusions.

Question 9

Where do DCs come from?

Answer 9

• HSC from BM

Macrophage-DC precursor –> Monocytes –> DCs and macrophages.

DCs can develop from monocytes

Question 10

Can you produce specific cells from the BM?

Answer 10

Yes! If we give HSC specific factors, they will differentiate into specific wanted cells. Nice!

Question 11

What factor will make HSC differentiate into DCs?

Answer 11

GM-CSF

Question 12

What kinds of endocytosis do we have? And what are they?

Answer 12

1) Phagocytosis - Big particles through receptor recognition. Wraps around the particle and needs receptor all along the particle (such as Ab binding for example).
2) Macropinocytosis - Membrane protrudes out into the area and takes in a big bud of the EC fluid with it’s included content.
3) Micropinocytosis - Inwards budding of the membrane with any content.

Question 13

TLR can respond two places with two responses. Which? What is this called?

Answer 13

Compartmentalised Signalling

• Respond from plasma membrane (increase macropinocytosis “sample from surroundings” + pro-inflammatory cytokines) and from endosome (increase in pro-inflammatory cytokines + IL-12).

Question 14

What do antigen-capture lead to?

Answer 14

• An increase in endocytosis like macropinocytosis.

That is. Stimulate with LPS and cells will increase endocytosis.

Question 15

Which receptor is responsible for increased endocytosis after antigen-capture?

Answer 15

• TLR2 and TLR4

Question 16

Explain what we can see with an ELISA and the basic principal

Answer 16

• We can see cytokine produced by the cells. In exercise 1 we used sandwich ELISA (the far most used one)

Question 17

Explain a sandwhich ELISA

Answer 17

1) Plate the plate with target-specific Ab - “Capture Ab”
2) Add sample with protein off interest
3) Add another mAb that binds to another epitope on the same protein of interest.
- This Ab is bound with markers such as Biotin (can bind streptavadin)
4 A) Add secondary antibody with conjugate OR
4 B) Add Streptavadin-HRP complex (Horseraddish peroxidase). The Streptavadin binds strongly to Biotin. Add substrate to the HRP which will make a blue product that can be measure with Optical Density (OD) and concentration measured through a standard curve.

I vores forsøg var Ab der bandt efter capture antibody direkte conjugeret til Biotin, som kunne binde streptavidin-HRP complekset.

Question 18

Explain the three types of ELISA with their strengths and weaknesses

Answer 18

1) Direct Assay - Coat plate with Antigen - add Ab-enzyme complex that binds to the Antigen –> Add substrate –> OD
- Make specific Ab for each Antigen, less specific, high background noise from other proteins and off target bindings.
2) Indirect Assay - Coat plate with antigen - Add primary Ab that binds antigen, add secondary ab-enzyme complex –> Add substrate –> OD
- Amplification through secondary Ab, potential cross-reactivity by secondary Ab
3) Sandwhich ELISA - Coat plate with Ab - Add Antigen - incubate with Ab - add Ab-enzyme or streptavadin-enzyme complex - add substrate - measure OD
- Can increase sensitivity. Can used same Ab for last step with the enzyme.

Question 19

Explain FACS (Fluorescence-activated Cell Sorting)

Answer 19

Ligesom flow cytrometry, men med forskelligt farvede lasere der baseret på deres readout af intensiteten kan sortere cellerne. Vi kan derfor staine/farve et overflade protein og sortere celler efter dette.

Question 20

Hvad ser vi i vores resultater fra forsøg 1?

Answer 20

• Da vi forsøger at differentiere BM til DCs forventer vi at se vores flow cytometry at celle antallet i vores P2 gate, som markerer runde celler, bør falde. Det gør det også. Helt perfekt.
• Vi ser dog stadigvæk mange celler i P2 gaten (runde og små), derfor er ikke alle cellerne differentierede. De kan allrede have valgt lineage (eks. erythrocytter)
• Inden differentiering ser vi både små, runde celler og mellemstore granulære celler som kan være neutrophiler eller deres precursos.

Question 21

I forsøg 1, hvor ville lymphocytter placeres i vores flow cytometry?

Answer 21

• De er små og runde og ville derfor være i P2 gaten, som vi ser det i de givne spleen data, sammen med eks. erythrocytter og hematopoietiske stamceller (ikke I spleen!).

Question 22

Hvad var målet med forsøg 2?

Answer 22

3 mål: Cytokine respons, signaleringsmolekylær og endocytose.

• Undersøge innate signalering i umodne BM dereivierede DCs ved at kigge på cytokine responset på 3 stimuli: Gram positiv bakterier, Pam3CSK4 og Peptidoglycan (to pathogen-associated molecular patterns (PAMPs)
• For derefter at inhibere essentielle signalleringsfaktorer, NF-kB og MAP kinaserne P38 og JNK for at se hvilke der er involverede i signal transductionen ved forskellig stimuli.
• Til sidst kigger vi på phagocytose ved optag af en fluorescent labelled bakterie og macropinocytosis ved optag af fluro labelled dextran.

Question 23

Hvilke 3 hovedcelletyper kan lave APC og phagocytose?

Answer 23

I rækkefølgen af mest APC:

1) DC
2) Macrophager
3) Neutrophiler

I rækkefølgen mest nedbrydende:

1) Neutrophiler
2) Macrophager
3) DC

Question 24

Hvilken rolle spiller cytoskelettet for endocytose?

Answer 24

• Det kræver omraarangering af cytoskeletet således at membranen kan omslutte/protrude eller “indslutte”. Vi skal altså bruge aktin og mikrotubuli aktivitet!

Question 25

Hvilken pathway signallerer både NOD receptors, TLR, IL-1 receptors and TNF receptor igennem?

Answer 25

• MAP Kinase subtyperne, c-Jun, ERK og p38

- Essentielle pathways for initiering og regulering af ekspressionen af immun mediatorer.

Question 26

Hvilken alternativ pathway kan TLR lede til?

Answer 26

• Produktionen af Interferon Respons Faktorer (IRF)

Question 27

Hvad genkender TLR?

Answer 27

• Et udvalg af bakterielle komponenter så som Lipopolysakkarid (LPS) eller Lipoteichoic acid (LTA)

Question 28

Hvad sker der ved TLR aktivering?

Answer 28

1) Binding af ligand til homo-/hetero- TLR dimer
2) TLR danner Tyrosine Immune Receptor (TIR) motiver
3) Adapter proteiner binder til TIR vha af deres egne TIR motiver.
4) To typer af adapter proteiner kan nu binde: Cytokine inducerende (MyD88) eller sorterende adaptor proteiner (TRAM) som afgør om signallering finder sted i plasmamembranen eller i den endosomeale membran.

Question 29

Hvor er NOD receptors lokaliserede? Hvad genkender de?

Answer 29

• I celle cytosolen.
• Genkender nedbrudte mikrobielle komponenter og aktiverer et pro-inflammatorisk respons med sekretion af pro-inflammatoriske cytokinersåsom IL-8, IL-1beta, IL-23

Question 30

Hvad binder IL-1 receptoren og hvad aktiverer den?

Answer 30

• IL-1beta

- Indeholder TIR domæner og aktiverer samme pathway som TLR

Question 31

Hvordan blev DC “renheden” afgjort inden forsøg 2 gik igang?

Answer 31

• Vha. en farvning med en anti-CD11c markør og flow cytometry.

Question 32

Hvorfor bruger vi PAM3CSK4 til at stimulere TLR2 og TLR1?

Answer 32

• En analog til lipopretiner der er tilstedet i mange gram positive bakterier.
• Fordi en specifik peptidoglycan receptor ikke er endeligt bevidst endnu, man tror derfor det er et senere nedbrydningsprodukt der stimulerer TLR.

Question 33

Hvad er CytD?

Answer 33

• En reversibel inhibitor af aktin polymeriseringen - dvs. den ikke nødvendighvis hæmmer hele polymeriseringen.

Question 34

Hvad ser vi af cytokine produktion i DCerne ved stimulering med hhv. bakterien, peptidoglycan og PAM3CSK4?

Answer 34

• Bakterie: Lille sekretion af IL-12, men tæt på ingenting af IL-1B og IL-23
• PAM3GSK4 + Peptidoglykan (PGN): Stor IL-12 sekretion + sekretion af både IL-23 og IL-1B

Question 35

Hvilke cytokiner kigger vi på i Exercise 2?

Answer 35

IL-12, IL-1B og IL-23

Question 36

Stimulering af TLR2/TLR4 giver hvilken funktion i plasmamembranen vs. endosome?

Answer 36

Plasmamembranen: Øget macropinocytose + pro-inflammatoriske cytokiner.

Endosomet: Øget IL-12 + pro-inflammatoriske cytokiner.