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

Question 1

Tumor vaccination can be ex vivo/conventional vaccination or in vivo/in situ vaccination. Explain the process of conventional vaccination.

Answer 1

In ex vivo/conventional vaccination an identified tumor antigen is used together with an adjuvant. It is then systemically injected into the body where it will be taken up by APCs which causes activation and expansion of effector T cells that recognize the specfic vaccine antigens and can fight the tumor.

Question 2

Tumor vaccination can be ex vivo/conventional vaccination or in vivo/in situ vaccination. Explain the process of in vivo/in situ vaccination.

Answer 2

Here, the vaccine only consists of an adjuvant. The vaccine is injected into the tumor (intratumoral injection). The adjuvants boosts the immune system and exploits all relevant tumor antigens avaible in the tumor. This causes activation and expansion of effector T cells that can recognize all relevant tumor antigens.

Question 3

Oncolytic viruses (like the adenovirus) can be used in treatment of cancer. Explain how an oncolytic virus can kill cancer cells but keep normal cells intact.

Answer 3

Oncolytic viruses are molecularly changed so that they cannot divide in normal cells, but can in cancer cells. This is because cancer cells have activated pathways that protects them against apoptosis. Oncolytic viruses can replicate in cancer cell and upon virus release the cancer cells gets destroyed (lysis). The new virus particles can infiltrate new cancer cells and kill these as well.

Question 4

How are oncolytic virusses also able to activate an anti-tumor immune response?

Answer 4

With the lysis of cancer cells and the release of virus particles, cancer antigens will be released that activate the immune system. They also carry TLR ligands that can help in activation of dendritic cells.

Question 5

What’s the difference between active and passive immunotherapy?

Answer 5

The aim of active immunotherapy is to stimulate the host’s immune system or a specific immune response to a disease/pathogen (most used in cancer treatment). The aim of passive immunotherapy is to boost and help the immune system in fighting off an infection or disease, this is usually done with the help of antibodies.

Question 6

Steve Rosenberg became known for his treatment against melanomas. What kind of immunotherapy did he came up with (active/passive) and how did he came up with this treatment?

Answer 6

He came up with passive immunotherapy –> vaccination with in vitro primed/expanded T cells. He excised tumors from a patient which he fragmented. The fragments of these tumor were then cultured in the lab with IL-2 (growth factor for T cells) which caused T cells to activate and proliferate. The T cells were then examined whether they could recognize the specific tumor antigens and where then put back into the body.

Question 7

What was the thing that at last made the immunotherapy of Steve Rosenberg work?

Answer 7

Before injections of the tumor antigen specific T cells, the patients get treated with standard chemotherapy or radiation (lymphodepleting chemotherapy). This causes complete destruction of (among others) T cells. When the tumor antigen specific T cells are injected, they don’t need to compete with other ‘normal’ T cells and thus make them more effective.

Question 8

What treatment is there if you have a tumor that’s not operable (so it’s not possible to isolate tumors, like in the therapy of Steve Rosenberg)?

Answer 8

CAR T cell therapy

Question 9

CAR T cells stand for Chimaeric Antigen Receptor T cells. They are used in CAR T cell therapy. How are CAR T cells generated?

Answer 9

In this treatment antibody fragments that bind to specific antigens are fused to the end of a T cell receptor (TCR). The antibody is transduced into the T cell with the help of a retrovirus.

Question 10

How does CAR T cell immunotherapy work?

Answer 10

Blood is extracted and leukopheresis is performed. You grow out the T cells with the help of IL-2. Through retroviral transduction anti-CD19 CAR (antibody) is transduced into the T cells. Lymphodepleting chemotherapy is performed on the patients and the anti-CD19 CAR T cells are injected into the body. And when this complex recognizes the specific tumor antigens that anti-CD19 CAR can bind, the T cells become active.

Question 11

How can inflammation indirectly cause DNA damage?

Answer 11

Injury/irritation/infection will result in an inflammatory reaction (recruitment of mast cells, neutrophils and monocytes). These immune cells respond with a respiratory burst (essential for e.g. degradation of pathogens) and release of free radicals. These can cause protein damage, lipid peroxidation and DNA damage and mutation.

Question 12

Immune cells interact with the tumor with the help of cytokines. Cytokines can have pro- and anti-tumor effects. Name cytokines that suppress tumor growth (functions will be discussed in detail later).

Answer 12

IL-12 (and IL-23)

Question 13

Immune cells interact with the tumor with the help of cytokines. Cytokines can have pro- and anti-tumor effects. Name groups of cytokines that progress tumor growth (functions will be discussed in detail later).

Answer 13

1. TNF-a, IL-6 and IL-10
2. TGF-b
3. TNF-a, IL-6 and TGF-b
4. TNF-a, IL-17 and TGF-b
5. TNF-a and TGF-b

(TNF-a, IL-6, IL-10, TFG-b and IL-17)

Question 14

The cytokine IL-12 suppresses tumor growth. How?

Answer 14

IL-12 activates NK cells and cytotoxic T cells.

Question 15

What is the function of TNF-a, IL-6 and IL-10?

Answer 15

They enhance tumor cell growth.

Question 16

The cytokine TGF-b enhance tumor growth. How?

Answer 16

It enhances tumor cell tissue invasion.

Question 17

The cytokines TNF-a, IL-6 and TGF-b enhance tumor growth. How?

Answer 17

They affect stromal cells and enhance metastasis.

Question 18

The cytokines TNA-a, IL-17 and TGF-b enhance tumor growth. How?

Answer 18

They affect endothelial cells and enhance aniogenesis.

Question 19

The cytokines TNF-a and TGF-b enhance tumor growth. How?

Answer 19

Affect NK cells, CTLs and macrophages and impair their function.

Question 20

What cytokine kan suppress antitumor immunity?

Answer 20

IL-10

Question 21

Inflammation or UVB light can damage the lipid bilayer of the cell membrane. What happens after damage to the lipid bilayer?

Answer 21

With damage to the membrane, phospholipases get activated. The phospholipases release arachidonic acid from the membrane and with the help of COX1 and COX2, cyclooxygenase is formed that can produce prostaglandins (PGE2, PGF2, PGI2).

Question 22

How do prostaglandins contribute to carcinogenesis?

Answer 22

They are pro-inflammatory and can drive tumor proliferation. They can also block dendritic cell development.

Question 23

Any inflammatory response is accompanied by feedback loops that lead to the generation of immune suppressive cells. This is a way of the body to try and prevent a chronic inflammation.

But in cancer this is a way for cancer to evade the immune system. In this pathway, tumor cells are involved, but also regulatory T cells and myeloid suppressor cells that all influence T cell function. What is the function of these cells which affect T cell function?

Answer 23

• Tumor cells produce high concentrations of TGF-b and IL-10.
• Myeloid suppressor cells (immature dendritic cells) release ROS and NO that deplete arginine concentrations vital for T cell proliferation.
• Regulatory T cells also produce high amounts of TGF-b and IL-10.

Question 24

What are the 3 E’s?

Answer 24

The 3 E’s stand for Elimination, Equilibrium and Escape. They describe how tumors evade the immune system (also called immuno-editing), which goes along the process of “survival of the fittest” (tumors that are best able to evade the immune system, are the cells that survive.

Question 25

What is elimination in immuno-editing?

Answer 25

Tumor cells don't spontaneously arise and grow. Elimination is the response of the immune system to the arising tumor cells by activation of all kinds of immune cells (NK, CD8+, CD4+, macrophages etc.) which results in the elimination of these tumor cells.

Question 26

What is equilibrium in immuno-editing?

Answer 26

Some tumor cells have gained mutations and have the ability to escape the immune response. So in equilibrium some cells can start to grow (the ones that can evade the immune system) and some get destroyed by the immune system.

Question 27

What is escape in immuno-editing?

Answer 27

Accumulation of mutations occur over time in the surviving cancer cells. This will in the end cause complete immune evasion of cancer cells (e.g. by antigen or MHC loss). Cancer cells can then invade and metastasize.

Question 28

The last flashcard mentioned 10 ways tumors evade the immune system. The last two (immunosuppression by tumor-derived factors and DC suppression/depletion + MDSC accumulation) are major problems and need new vaccination strategies to circumvent these problems. Why are they such a big problem?

Answer 28

A tumor suppresses the immune system by releasing tumor-derived factors that can suppress the immune system (like IL-10 and IL-6). When a tumor metastasizes these tumor-derived factors can become systemic and can (for example) widely suppress dendritic cells to become mature. This causes accumulation of myeloid dendritic suppressor cells (MDSC) that suppress T cell proliferation.

Question 29

Do you expect T cell tolerance and immune suppression in melanomas? Why?

Answer 29

Melanomas have a high somatic mutation rate and thus try the hardest to escape the immune system. In these tumors you can find loads of immune cells that all have a hand in suppressing the immune system (Treg, Th2, suppressive cytokines, mono- and granulocytic MDSC). Note that this phenomenon can also be found in draining lymph nodes.

Question 30

What is an important cause of therapy resistance?

Answer 30

Subverted myeloid cells that release pro-angiogenic and tumor invasion promoting factors.

Question 31

Tumors can suppress the immune system by downregulating MHC class I molecules. But cells that have downregulation of MHC class I can react to this to prevent immune suppression. What reaction is this?

Answer 31

NK cells become active (NK cells are important in killing of virusses who also have gotten better in downregulating MHC molecules).

Question 32

NK cells have a Killing Inhibitory Receptor (KIR) on their surface. It prevents them from killing random cells. To wat do KIRs need to be bound to in order to suppress NK cell activation?

Answer 32

To HLA-molecules. When these are present on cells, NK cells can't get activated for killing of cells. But when HLA-molecules disappear (like in cancer) the NK cell becomes active and is able to kill the cancer cells.

Question 33

How can a heterogeneous tumor with a HLA-negative tumor nest (due to β2m (constituent of MHC complex) mutation) result in a homogeneous HLA-negative metastasis?

Answer 33

The primary tumor consists of a few cells that are HLA-negative and thus have a β2m mutation and consists of cells that still express the HLA molecules (and thus no mutation). The primary tumor is therefore called heterogeneous. These cells can escape the immune system, have more ability to divide and thus can metastasize. The metastasis then consists of homogeneous HLA- negative cancer cells that all have escaped the immune system.

Question 34

There are different phenotypes for HLA class I loss in cancers. Describe the following phenotypes and keep in mind that an HLA molecule contains two different haplotypes of different subunits (Cw2 and Cw7 for example): 1. HLA total loss 2. HLA haplotype loss 3. HLA locus loss 4. HLA allelic loss 5. Compound phenotype 6. Unresponsiveness to IFNs 7. HLA class Ia +/- HLA class Ib +

Answer 34

1. HLA total loss: there's total HLA loss if all subunits of a HLA molecule have been lost 2. HLA haplotype loss: if there's haplotype loss this means that there's loss of alleles that are inherited together from a single parent. So this means that if you get Cw7, A2 and B7 from your mother and Cw2, A24 and B44 from your father, that there can be haplotype loss if Cw7, A2 and B7 are not present anymore. 3. HLA locus loss: this means that the genes located on the loci on both chromosomes are lost, for example loss of A2 and A24. 4. HLA allelic loss: HLA allelic loss is loss of one specific gene of one chromosome (for example only loss of B44). 5. Compund phenotype: a combination of HLA allelic/locus/haplotype loss. 6. Unresponsiveness to IFNs: tumor cells carry mutations in the IFN pathway that blocks upregulation of MHC molecules. 7. HLA class Ia +/- HLA class Ib +: a certain phenotype that can bind to c-myc on T cells and suppresses them.

Question 35

What can go wrong in antigen processing pathway (with the ideal goal to present an antigen through MHC molecules) what results in a downregulation of MHC molecules (please think of multiple reasons).

Answer 35

1. They can downregulate antigens that are not needed for tumor survival (antigen loss) and thus there's less antigen and therefore less MHC needed to present antigens. 2. There can be something wrong with the proteasome, so that there's no antigen that fits on the MHC molecule. 3. Due to deletions/mutations there's loss of MHC I heavy chain or loss of β2 microglobulin 4. Something wrong with transport/vesicle formation

Question 36

What is the difference between hard and soft MHC-I lesions?

Answer 36

Hard MHC-I lesions are genetic alterations leading to permanent loss of MHC-I. Soft MHC-I lesions are reversible e.g. by IFN-y stimulation

Question 37

What cells may help in complete loss of MHC molecules if T cells can't be activated?

Answer 37

CAR-T cells, because they carry an antibody fragment that instantaneously can recognize the surface of a tumor cell.

Question 38

T cells become active when an antigen is presented to them through an APC. But they can also be suppressed through interaction and binding of PD-1 on the T cell to its' ligand PDL-1 on the APC. What happens when PD-1 binds with its ligand PDL-1?

Answer 38

Intracellular cascade that will lead to the blockade of activation of the TCR and costimulatory molecules. This causes reduced TCR signaling, cytokine production and target cell lysis.

Question 39

How is CTLA4 on T cells able to block an immune response?

Answer 39

By competing for the binding to B7-1 (CD80) on the APC with CD28. CTLA4 has a higher affinity and outcompetes CD28 and thus blocks activation.

Question 40

PD-1 and CTLA4 drugs seem to work best in different stages of tumor progression. Why is CTLA-4 a target in **draining lymph nodes** and why is PD-1 (and PD-L1) a target in the **tumor environment**?

Answer 40

* Because CTLA-4 outcompetes CD28, CD28 is the first co-stimulatory molecule to be blocked. When this signal isn't blocked, it is important in initiating T cell response in the lymph node. Thus binding of CTLA-4 to B7, is the first to aid cancer in carcinogenesis. Therefore, targeting CTLA-4 in draining lymph nodes is most effective. * Binding of the ligands of PD-1 (and PD-L1) are responsible for inactivating effector T cells (T cells have already been primed and activated in lymph nodes). Therefore, targeting PD-1 and PD-L1 in the tumor environment, where T cell functions have been inactivated due to ligand and PD-1 interaction, is most effective.

Question 41

There are two ways PDL1 is expressed on tumor cells. What are these two ways?

Answer 41

Through either innate or adaptive immune resistance.

Question 42

How are tumor cells able to express PDL1 through the innate immune resistance pathway?

Answer 42

Through activation of oncogenic pathways, this leads to constitutive expression of PDL1.

Question 43

How are tumor cells able to express PDL1 through the adaptive immune resistance pathway?

Answer 43

When tumor cells with an antigen presenting MHC on their surface bind and activate T cells, the T cells will produce IFN-y. Through STAT signaling IFN-y expression can lead to PDL1 expression.

Question 44

Study this picture

Answer 44

Ok