Chapter 12 and 13: Immunology, immunotherapy and inflammation (Lecture 1/2, main)

Question 1

What is usually a big problem in treatment of cancer through the use of standard treatment options and is not (or less of) a problem for immunotherapy?

Answer 1

Resistance (the first 7 slides of the lecture introduces pembrolizumab, an anti-PD-1 antibody used against advanced melanoma. Previously ipilimumab was used against meloma, where a lot of patients still died due to resistance to the medicine).

Question 2

T cells express PD-1 (programmed cell death protein 1) and CTLA4. What is their function?

Answer 2

Both of these receptors on the T cells act as brakes in the regulation of the immune system.

• PD-1 is important in preventing autoimmunity by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity.
• CTLA4 functions as an immune checkpoint and downregulates the immune response.

Question 3

One of the hallmarks of cancer is avoiding immune destruction. Why and how are PD-1 and CTLA4 important immune escape mechanisms?

Answer 3

Because they regulate the immune response. They are important for preventing auto-immunity and thus are a useful tool for cancer cells to evade immune destruction. Cancer cells can express the ligands for CTLA4 and PD-1 to repress the immune system’s response.

Question 4

How can antibodies be used in the setting of cancer cells that repress the immune system by activating CTLA4 or/and PD-1

Answer 4

Antibodies that prevent interaction between CTLA4 and PD-1 and their ligands.

Question 5

If you immunize two mice with irradiated tumor cells and next up inject viable cells of the same tumor in mouse 1 and inject viable cells of a different tumor in mouse 2.

What will happen to mouse 1 and 2?

Answer 5

• Mouse 1 will react to the tumor with unique tumor rejection antigens which elimate the tumor/viable cells.
• Mouse 2: since this mouse has been immunized with another type of tumor (and thus injected with a different viable tumor) it can not eliminate the tumor.

Question 6

How does a cytotoxic T cell kill a virus infected cell?

Answer 6

It recognizes MHC class I in complex with a viral peptide and kills the infected cell.

Question 7

Explain in short how dendritic cells and T cells can kill tumor cells.

Answer 7

The tumor is recognized by dendritic cells which engulf particles of these tumor cells to present them to (among others) T cells in the lymph nodes. The T cells get activated and migrate to the tumor and then kill the tumor cells.

Question 8

What is the Mellman cancer immunity cycle?

Answer 8

It shows a proces through which immune cells eliminate tumors.

Question 9

There are 7 steps in the Mellman cancer immunity cycle. What are these?

Answer 9

1. Release of cancer cell antigens by a tumor
2. Cancer antigen presentation (to dendritic cells)
3. Priming and activation (of T cells)
4. T cells migrating/trafficking to the tumor
5. Infiltration of T cells into tumors (migrating from the blood vessel to the tumor)
6. Recognition of cancer cells by T cells
7. Killing of cancer cells by T cells

(please remember the fact that in the process of killing the tumor cells (step 7) new tumor antigens are being released (step 1) and so the cycle continues)

Question 10

Tumors have ways to evade this Mellman cancer immunity cycle. What would be the case for this in step 2 (cancer antigen presentation)?

Answer 10

That dendritic cells may not develop properly due to suppressing factors secreted by the tumors.

Question 11

Tumors have ways to evade this Mellman cancer immunity cycle. What would be the case for this in step 3 (priming and activation)?

Answer 11

That secreted factor by the tumor slow down the dendritic cells so much that T cells can never start to proliferate.

Question 12

Tumors have ways to evade this Mellman cancer immunity cycle. What would be the case for this in step 4 (T cells migrating to the tumor) and step 5 (infiltration of T cells into tumors)?

Answer 12

That tumor blood vessels are not properly functioning and don’t express the adhesion molecules that T cells need to adhere to blood vessels and to migrate out of them.

Question 13

Tumors have ways to evade this Mellman cancer immunity cycle. What would be the case for this in step 6 (recognition of cancer cells by T cells)?

Answer 13

That T cells interact with the tumor and the tumor puts a brake on the T cells (for example PD-1 which was previously discussed)

Question 14

It’s important to develop therapies that make sure T cells can do their job. Through what therapies can we make sure that tumor antigens are released (step 1) in a way that dendritic cells can become activated?

Answer 14

Chemotherapy, radiation therapy and targeted therapy

Question 15

What drugs can help dendritic cells to become active (step 2)?

Answer 15

Certain cytokines (IFN-a, GM-CSF, anti-CD40, TLR) (Will be discussed in detail later).

Question 16

What drug helps if dendritic cells in the lymph node cannot stimulate activation and proliferation of T cells (step 3)?

Answer 16

Anti-CTLA4 (also anti-CD137, anti-OX40, anti-CD27, IL-2 and IL-12)

Question 17

What is the function of anti-angiogenic drugs like anti-VEGF antibodies?

Answer 17

To stimulate tumor blood vessels to express the right adhesion molecules for T cells to enter the tumor (step 4 and 5).

Question 18

What drugs stimulate T cells to do their job in the tumor environment (step 7)?

Answer 18

Checkpoint inhibitors like anti-PD-L1 and anti-PD-1 (and IDO inhibitors)

Question 19

How is a CD8+ cell activated (with an intracellular/endogenous antigen)?

Answer 19

When there’s a virus or tumour antigen in cells, this foreign antigen gets degraded by a proteasome. The antigen is cut into peptides and transported to the ER. In the ER MHC class I molecules will be formed that can bind the antigenic peptides and translocates as a complex to the surface where it can interact and activate CD8+ T cells.

Question 20

How is a CD4+ cell activated (with an extracellular/exogenous antigen)?

Answer 20

An exogenous antigen is taken up by an antigen presenting cel (APC) through an fagosome which fuses with granules to degrade the antigen (fagolysosome). In the ER MHC class II is produced along with another protein (CLIP) that occupies the peptide binding domain of MHC molecule. It is then transported and fused with the fagolysosome that contains the antigenic peptides. The complex with MHC-peptide is brought to the surface where it can activate CD4+ T cells.

Question 21

Why does the protein CLIP occupy the peptide binding domain of MHC II molecules?

Answer 21

Only antigenic peptides can outcompete CLIP. Thus, it’s a mechanisms that prevents other peptides from binding to MHC II molecules and thus makes sures that only antigenic peptides can bind.

Question 22

So normally endogenous antigens will bind to MHC-I molecules and exogenous antigens will bind to MHC-II molecules. But there’s a way for exogenous antigens to present themselves via MHC-I molecules, through cross-presentation. This pathway is important for initiating a cancer immune response since cancer antigens will be taken up by APCs by phagocytosis. Their are two pathways: the cytosolic and vacuolar pathway. Explain the vacuolar pathway.

Answer 22

First the exogenous antigen (e.g. cancer antigen) is taken up via phagocytosis.

• the vacuolar pathway simply states that in phagosomes sometimes MHC class I molecules will be present where through phagosomal degradation, antigenic peptides can be bound to MHC class I and can be presented on the cell-surface.

Question 23

So normally endogenous antigens will bind to MHC-I molecules and exogenous antigens will bind to MHC-II molecules. But there’s a way for exogenous antigens to present themselves via MHC-I molecules, through cross-presentation. This pathway is important for initiating a cancer immune response since cancer antigens will be taken up by APCs by phagocytosis. Their are two pathways: the cytosolic and vacuolar pathway. Explain the cytosolic pathway.

Answer 23

First the exogenous antigen (e.g. cancer antigen) is taken up via phagocytosis. Next the antigen will be transported from the phagosome to the cytosol where it will be degraded via a proteasome. From then on it two things can happen:

• the antigenic peptides will be transported into the ER, where MHC class I is produced and where MHC I and the antigenic peptide bind. The complex is transported to the cell-surface for antigen presentation.
• the antigenic peptide is tranported into a phagosome where MHC class I is also present and will bind with eachother. The complex is transported to the cell-surface for antigen presentation.

Question 24

T cells need 3 signals to become active. Shortly describe these steps.

Answer 24

1. APC (dendritic cell) with MHC molecule(+antigen) binds to TCR of T cell.
2. Costimulation -> CD80 on APC can interact with CD28 of T cell and CD40 on APC can interact with CD40L
3. Th differentiation (cytokines released from APC that can bind to T cell receptor for differentiation).

Question 25

Fill in: If the dendritic cell secretes IL-12 for signal 3 (Th differentiation) the naive T cell can differentiate into Th...(1) and can secrete factors like ... (2) and ... (3). These cells can help when there's a ... (4) infection or a ... (5). If a dendritic cell secretes IL-4 for signal 3 (Th differentiation) the naive T cell can differentiate into Th... (6) and can secrete factors like ... (7) and ... (8) which are important for antibody activation.

Answer 25

If the dendritic cell secretes IL-12 for signal 3 (Th differentiation) the naive T cell can differentiate into **Th1 cell** and can secrete factors like **IFN-y** and **TNF-b**. These cells can help when there's a **viral** infection or a **tumor**. If a dendritic cell secretes IL-4 for signal 3 (Th differentiation) the naive T cell can differentiate into **Th2 cell** and can secrete factors like **IL-4** and **IL-8** which are important for antibody activation. 1. Th1 cell 2. IFN-y 3. TNF-b 4. viral 5. tumor 6. Th2 cell 7. IL-4 8. IL-5

Question 26

Name the function of Th1, Th2, Treg and Th17 cells.

Answer 26

- Th1: important in cellular immunity and clearance of intracellular pathogens - Th2: important in humoral immunity, clearance of extracellular pathogens and allergy - Treg: important in tolerance and immune suppression - Th17 cells: important in inflammation, autoimmunity, clearance of certain extracellular pathogens.

Question 27

Why is the differentiation into Th1 cells good news in the fight against cancer but differentiation into Th2, Treg or Th17 not?

Answer 27

- Th1 cells secrete factors that are important for clearance of cancer cells. Th2 cells can also help in this fight, but don't do as good of a job as the Th1 cells. - Also if Th2 cell are dominant over Th1 cells, they can have a suppressing influence over Th1 cells. - Presence of Treg can induce immune suppression/tolerance against the tumor. - Th17 cells can be good or bad. It's responsible for tissue inflammation which can be good in some situations in the fight against cancer but bad in others.

Question 28

In general this picture has now been discussed (except for a few things like NK cells and plasma cells that can also help the immune system with fighting cancer). Please look at this picture and ask yourself if you know/understand everything.

Answer 28

Ok

Question 29

How does the release of cancer antigens result in activation of dendritic cells and how do dendritic cells become active?

Answer 29

Dendritic cells (DCs) reside in peripheral tissues are in an immature resting state (when there's no danger) and express all sorts of molecules that they need to bind pathogens. When danger (bacteria, virus, cancer, tissue damage) is present and pathogens are bound/engulfed by the DC, the DC becomes activated and gets transported to lymphoid tissue. In this active state, the DC will express costimulatory signal needed to activate T cells.

Question 30

Toll-Like Receptors (TLRs) are promising adjuvants in cancer therapy. What are Toll-Like Receptors?

Answer 30

Receptors on the surface of APCs (or in endosomes) that can bind different ligands derived from pathogens.

Question 31

# Fill in: Extracellular Toll-Like Receptors (TLRs) mostly bind.... (1) while intracellular TLRs mostly bind.... (2).

Answer 31

1. Bacteria and yeast 2. DNA/RNA derived from intracellular virusses or bacteria. (antigenic tumor cells can also bind to TLRs)

Question 32

What happens after pathogens bind to Toll-Like receptors?

Answer 32

They activate a **signaling cascade** which goes through **MyD88**(!) that can bind to the **cytoplasmic tail** of these TLRs. When they bind their ligands, these become **phosphorylated** where the signals moves down into the **nucleus** where a proper response occurs. This can either be an **antiviral immune repsonse, proper T cell stimulation** or **stimulation of inflammation**.

Question 33

What is the STING (stimulator of interferon genes) pathway?

Answer 33

This pathway recognizes cytosolic double-stranded DNA derived from our own cells (like tumor cells that are dying) and produced IFN-molecules as a response.

Question 34

Describe the STING pathway.

Answer 34

* When dsDNA is present in the cytosol it interacts with cGAS (cyclic GMP-AMP Synthase). * cGAMP is formed (intracellular cyclic dinucleotides) that act as second messengers. * This leads to the aggregation of STING in the ER. * This causes TBK1 (tank-binding kinase I) to be phosphorylated and activated. * This causes phosphorylation of of transcription factor IRF3 (interferon regulatory factor 3). IRF3 stimulates the transcription of type I interferon (IFN) genes. * These genes can stimulate cytotoxic T cells to become active and dendritic cells to become mature.

Question 35

Describe how endogenous type I IFN signaling (STING pathway) is required for intratumoral accumulation of (cross-priming) DCs.

Answer 35

A dendritic cell takes up antigens of the tumor where the dsDNA is recognized by the STING pathway. This leads to production of (among others) IFN-b. IFN-b can activate other dendritic cells. Active dendritic cells can cross-present tumor antigens to T cells to induce activation into naive CD8+ T cells that become effector CD8+ T cells (or cytotoxic T cells). Effector T cells in turn secrete IFN-y to kill tumor cells.

Question 36

Tumor antigens can arise through different mechanisms. Name four of these mechanisms.

Answer 36

1. Overexpression of certain genes (like EGFR) 2. Expression of tissue-specific proteins on a tumor that normally only is expressed on specific tissue (like tyrosinase) 3. Expression of genes that normally are only expressed during embryogenesis, these proteins can be found in gonads but then they don't have HLA (these antigens are called cancer testis antigens). 4. Mutation of a gene results in a different gene product --\> new antigen (neoantigens)

Question 37

What can you say about the central tolerance of the antigens formed by these 4 mechanisms discussed above? Are all types of antigens tolerated by the immune system or are certain antigens/mechanisms more vulnerable to the immune system?

Answer 37

- Antigens that arise through mutation are very tumor specific and thus the immune system doesn't have any central tolerance. - The immune system has a low tolerance against cancer testis antigens (that arise due to activation of genes that are only needed during embryogenesis). - The immune system has a very high tolerance against proteins (and thus antigens) that are actually tissue-specific or are overexpressed, since these proteins are also present in normal healthy cells.

Question 38

Explain this figure.

Answer 38

This figure displays tumor types and their somatic mutation frequencies. This means that cancers like melanomas, lung and bladder cancers also respond the best to immunotherapy since their amount of neoantigens is highest. This is opposite with cancers like pancreatic cancer that have a much lower somatic mutation frequency and thus a lower response to immunotherapy.

Question 39

What is the definition of hot and cold tumors?

Answer 39

Hot tumor are tumors like melanomas, with a high somatic mutation rate. Cold tumors are like pancreatic cancer, with a low somatic mutation rate. This also means that there are more T cells found in hot tumors than in cold tumors, due to the non-existent tolerance of the immune system for the neoantigens.

Question 40

Provenge is a vaccine/treatment for prostate cancer. It contains fusion protein build out of a tumor antigen and a cytokine. What specific tumor antigen and cytokine(+function)?

Answer 40

It contains the tumor antigen PAP and the cytokine GM-CSF that can mobilize and activate dendritic cells.

Question 41

How does Provenge work?

Answer 41

Blood is taken from the patients where the fusion protein Provenge is co-incubated with. Then monocytes in the blood take up to fusion protein and differentiate into lookalike "dendritic cells". They can then take up the antigen and present it to cytotoxic T cells who can then interact and kill prostate cancer cells.