Lecture 10b

Question 1

What does each B cell produce?

Answer 1

Each one makes a single type of antibody that gets planted in the B cell’s membrane with the sticky surface facing outwards.

Question 2

Do most antibodies encounter an antigen that they bind to?

Answer 2

No, most antibodies never encounter an antigen that they can bind.

Question 3

When an antibody binds an antigen, what does this signal the B cells to do?

Answer 3

The B cell proliferates in which it tries to produce mutated antibodies that will be better at binding antigens than the original one.

Question 4

Do B cells generally proliferate into B cells with stronger binding antibodies?

Answer 4

Most of the time, no.

Question 5

What happens after the B cells have deliberately mutated and proliferated?

Answer 5

The B cells with the highest affinities give rise to plasma cells and memory B cells.

Question 6

What happens to proliferated B cells that don’t bind the antigen good?

Answer 6

They commit suicide by apoptosis.

Question 7

What are memory cells?

Answer 7

Antibodies in the cell membrane acting as receptors for when you get infected again. They “remember” the pathogen that infected your body the first time.

Question 8

What are plasma cells?

Answer 8

Smaller cells that secrete antibodies into the blood. They are missing their transmembranes, which is why they are small.

Question 9

What do the antibodies produced by plasma cells do?

Answer 9

They find the pathogens and “tag” them so that macrophages will come and engulf the pathogen.

Question 10

What do macrophages contain that is important? What do they do?

Answer 10

Macrophages have lysosomes inside of them, which contain digestive enzymes. When the macrophage engulfs the pathogen, the pathogen is fused with the lysosome so that the digestive enzymes break down and kill the pathogen.

Question 11

What is special about cancer cells antigens compared to normal cells?

Answer 11

Cancer cells present antigens in their outer membranes that normal cells don’t have.

Question 12

What does CAR-T technology do?

Answer 12

We create CAR-T cells which will attack cells bearing the cancer-specific antigens.

Question 13

What is step 1 for producing CAR-T cells?

Answer 13

Obtain a plasma cell line that produces an antibody that is capable of recognizing a cancer antigen.

Question 14

How do we obtain a plasma cell line that produces only the antibodies of interest (monoclonal antibodies)?

Answer 14

1) We inject a mouse with an antigen, which will be seen as foreign.
2) An immune response will be mounted against this antigen. We want to harvest the B cells from this.
3) We need to mix these B cells with cancerous plasma cells that can recognize the cancer antigen but do not produce the antibody. A protein is added to fuse the cells.
4) Then, we plate out the fused cells and see what antigens the antibodies bind to. We make more of the one that binds to our antigen of interest.

Question 15

Why do B cells need to be fused with the cancerous plasma cells?

Answer 15

Our B cells on their own do not recognize cancer cells because it just appears like one of our genes is being encoded for a lot. It is not seen as foreign.

Question 16

When do B cells divide?

Answer 16

They ONLY divide when an antigen is bound, otherwise, they do not divide.

Question 17

Once we believe we have fused B cells to cancerous plasma cells, what do we add to them?

Answer 17

We add a chemical (HAT) which will kill any non-fused cells. The cells that are fused will survive.

Question 18

What are hybridomas?

Answer 18

B cells fused to cancerous plasma cells that will secrete antibodies into the blood. This is as opposed to the antibodies just being on the plasma membrane.

Question 19

Once we have obtained hybridomas, what do we do with them?

Answer 19

We want to plate out the hybridomas at ‘limiting dilution’. This just means the cells are plated on multi-well plates so that different cells producing different antibodies will each be in their own well.

Question 20

What are clonal populations?

Answer 20

All cells in a compartment/well are derived from one cell.

Question 21

After we have multi-well plated our cells, what do we need to add?

Answer 21

We add our antigen of interest so that we can identify the cells making the antibody that will bind to them. The antigen remains attached to the compartment/plastic surface, so that the antigen will stick along with the attached antibody.

Question 22

After the antigen has been added to the multi-well plate, what do we do?

Answer 22

We wash the wells out so that the only thing remaining is the antigen of interest and the antibody that attached to it.

Question 23

How do we identify that the antibody has attached to the antigen of interest in our wells?

Answer 23

We add in a secondary antibody that fluoresces. This antibody will bind to the primary antibody and show our antibody that bound to the antigen of interest.

Question 24

Once our secondary antibody has shown us the primary antibody, what do we know? Then, what do we do?

Answer 24

This is the cell line that we want because it produced the primary antibody that latched onto our antigen of interest.

We can then grow a bunch of these cell lines to produce these antibodies.

Question 25

What is step 2 for producing CAR-T cells?

Answer 25

We clone the antibody genes from the cell line we want. This is usually done by RT-PCR.

Question 26

What is step 3 for producing CAR-T cells?

Answer 26

We make a gene that just encodes the sticky surfaces of both the heavy and light chains. This is the area that binds to the antigens and is called the chimeric antigen receptor.

Question 27

What are T cells?

Answer 27

Cells that produce T cell receptors which are similar to antibodies.

Question 28

What are 2 ways in which B cells and T cells differ structurally?

Answer 28

1) T cells have alpha and beta chain locuses that are the same size 2) The receptor is more complicated.

Question 29

Are T cell antibodies made by VDJ recombination like B cell antibodies?

Answer 29

Yes!

Question 30

What does the binding of an antigen to the T cell receptor do?

Answer 30

It activates the immune system in multiple ways that will strengthen the immune response.

Question 31

What cell does T cell receptor stimulation activate the production of?

Answer 31

Plasma cells which will secrete more antibodies.

Question 32

Which are more important in the immune response: B cells or T cells?

Answer 32

T cells are actually more important in the immune response because they make B cells more active and they prevent autoimmunity.

Question 33

What is step 4 of producing CAR-T cells?

Answer 33

We need to obtain T cells to make the CAR-T cells.

Question 34

How do we obtain T cells that can be used for CAR-T cells?

Answer 34

1) We need to remove blood from a patient and purify it to get T cells. 2) We take these T cells and insert the gene for CAR (chimeric antigen receptor). 3) We grow a bunch of these transgenic T cells. 4) We infuse these cells into the patient. These cells will then bind to cancer cells and kill them.

Question 35

What have we been able to successfully do with CAR T-cell therapies?

Answer 35

CAR T-cell therapies have been developed that successfully target antigens commonly found in blood cancers.

Question 36

What is an area that we have been unsuccessful with in CAR-T therapy?

Answer 36

1) Efforts to identify antigens that are on the surface of solid tumors but not healthy cells have been unsuccessful. The surrounding environment of solid tumors makes this difficult because it can be a physical barrier that prevents the infused CAR T cells from reaching tumor cells.