Exercise 3 - Post-transcriptional regulation of NKG2D-ligands

Question 1

NKG2D i what kind of receptor? On which cell does is function?

Answer 1

• A activating receptor found on NK Cells, CD8+ T cells, NK-T Cells, CD4+ T cells and other T cells.

Question 2

Mention a NKG2D ligand

Answer 2

• MICA/B

Question 3

When are NKG2D ligands upregulated?

Answer 3

They are upregulated due to cell stress (infection, DNA damage, miRNA, Heat Shock, TLR Signalling etc.)

This way, cells can show if they are stressed and be recognized to be elminated.

Question 4

What happens if we have to much or too little NKG2D ligand?

Answer 4

Too much NKG2D-ligand: Autoimmunity

Too little NKG2D-ligand: Cancer or persistent infection

Question 5

Mention the two families og NKG2D ligands

Answer 5

• MHC class I Chain-related A/B (MICA/B)
• ULBP: UL16-binding proteins (ULBP1-6)

MICA/B shows the biggest polymorphisms

Question 6

Hvad sker der når MICA ikke får dens PTM N-glykosylering?

Answer 6

• MICA/B transporteres ikke til cellemembranens overflade –> Nedreguylering i aktivering af NK og CD8+ T celler.

Question 7

Hvad er N-glykosylering vigtig for?

Answer 7

• Protein foldning

- Transport til cellemembranen

Question 8

Hvor ser vi ofte ændret N-glykosylering, og hvorfor?

Answer 8

• Vira og cancer

- Mindre NKG2D ligand til overfladen –> mindre chance for at blive opdaget.

Question 9

Hvor mange glykosyleringssites har MICA? Hvilket er det vigtigste?

Answer 9

8 potentielle N-glykosyleringssites!

• Surface expression is dependent on Asn8 site sammen med Thr24

Question 10

Hvad undersøgte vi i forsøg 3?

Answer 10

We transfected cells via electroporation using two different MICA constructs (one with N-glyco sites, and one without) we investigated whether N-glycosylation is important for the cell-surface expression of MICA, (hint: it is!) using flow cytometry and western blotting.

Question 11

Explain flow cytometry with and without antibodies. And explain FACS.

Answer 11

• Basic principle (without Ab): Cell solution passed through a tube/stream one cell at a time. Laser from “in front” of the sample passes through the cell. Side scatter (a measurement for granularity, as granules scatter the light to the side) is measured 90 degrees to the side, while forward scatter is measured directly behind the laser. SSC = Sidescatter, FSC = Forward scatter. FSC-A = Area, FSC-H = height.
• With Abs + FACS: Same principle, but each cell is tagged (or not) with a fluorescent antibody. This can then be measured through excitation by lasers. This way you can measure cells with a specific surface marker and MAYBE a cell’s production of a protein with GFP:protein construct or similar.

FACS: Flow with Ab where cells are sorted through charging the droplets and sorting with these charges.

Question 12

Where are certain immune cells such as lymphocytes, monocytes and granulocytes on a SSC vs. FSC flow cytometry plot?

Answer 12

• Lymphocytes: Small and round and not particularly granular (bottom left corner is)
• Monocytes: Bigger and slight more granular then lymphocytes (I.e. more to the right and slightly higher)
• Granulocytes: Size as monocytes circa, but much higher placed because of their granularity.

Question 13

What can you detect with a western blot? Describe a western blot

Answer 13

• Detect Specific proteins in a sample of tissue homogenate or extract

1) Kør gelelektroforese
2) Transfer til membrane i en “elektorde” sandwhich
3) Block membranen (forhindrer uspecifik binding)
4) Inkuber m. primært Ab
5) Vask
6) Inkuber m. sekundært Ab
7) Vask
8) Fremkald evt. via fluorescens.

Question 14

Hvad bør man altid inkludere på et Western blot?

Answer 14

• Ladder

- Loading control

Question 15

How are the constructs in exercise 3?

Answer 15

• A MICA construct with or without N-glycosylations sites. With N-terminal linked GFP and Myc tags.

Question 16

What can we tell from our Western blot?

Answer 16

• We expect to see GAPDH in all the wells, since its a household gene, and a positive, loadin control for our wells.
• We expect our MICA to be higher in the gel since it should have N-glycosylation and the MICA-mut to be lower in the gel (wandered further) because of the lack of N-glycosylation and therefore a lower weight.
• Lastly, the western blot, confirms whether or nor our cells were successfully transfected. If we see a GFP-MICA-complex (with our without N-glycosylation), we know that the transfection was successful, which is important for the flow cytometry.

Question 17

What is the pUC18?

Answer 17

• In this case, an empty vector for control (negative control), to check if the transfection had any effect on the production nog N-glycosylation of GFP. Has an antibiotic resistance gene inserted.

Question 18

What can we tell from the flow cytometry analysis?

Answer 18

• Electroporation transfection has a negative impact on viability (much lower numbers in transfected cells)
• No GFP or MYC signal in Non-transf. cells as expected (neg. controls).
• Same percentage GFP positive cells in MICA and MICA-mut
• While Myc positive cells where much lower in MICA-mut cells, meaning the MICA complex is not transported to the membrane in MICAmut cells, but is detected IC through the GFP tag.

Question 19

Why do we use anti-Myc antibody for detection of MICA by FACS and anti-GFP for detection of MICA on WB?

Answer 19

1) GFP-tag could possibly not be expressed on the EC part of MICA/surface of cell = unfit for FACS, but the Myc-tag could in contrast be in the extracellular part of MICA. Doesn’t matter in WB, cells are lysated. (Either are expressed EC according to Søren though)
2) Trial and error proved the two tags to be good for each testit to be this way.
3) Myc-tag helps us differentiate between endogenic MICA and transfected MICA. So we couldn’t just stain for the endogenic MICA.
4) SDS vs. native protein structure could also play a role.

Question 20

Is N-glycosylation important for the regulation of MICA?

Answer 20

YES. Without it, MICA isn’t sent to the plasma membrane –> less killing and easier tumour growth.

BUT there is a type MICA008 der ikke har nedreguleres ved Asn8 fjernelse som ellers.

Question 21

Theoretically, how will the conclusion from these data impact a cancer cell without N-glycosylation modifications?

Answer 21

Without transportation to the cell membrane, immune cells cannot bind MICA, this NKG2D-ligand on the cell surface.

• -> NK cells or CD8+ T-cells will therefore not kill an infected cell or a cancer cell.
• -> increases invasiveness and metastasis of the cancer.
• -> A cancer with a normal N-glycosylation of MICA will not be able to thrive as well, as NK cells and CD8+ T-cells will recognise the NKG2D ligand and kill it.

Question 22

Hvad er de 3 vigtige ting når man bruger Antibodies i et forsøg?

Answer 22

1) Titrer dit antibody ned. Ved for høje conc. får du binding over det hele grundet non specific binding.
2) Brug en isotype control
3) Vær opmærksom på Fc receptorer (som hos NK celler) - block enten med et anti Fc Ab eller tilføj en masse ikke fluoscerende Ab til at optage dem.