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Biology 465 > Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression > Flashcards

Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression Flashcards

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1
Q

In this paper, where was the experiment taking place?

A
  • In the mice bone marrow microenvironment

- in vitro culture samples from humans

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2
Q

What did the addition of clodrolip result in?

A
  • TAMs began to die and released TNF and TRAIL
  • the dying TAMs resulted in the leukemic cells upregulating the expression of the TNF and TRAIL receptors
  • > death of leukemic cells
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3
Q

What type of leukemic cell did they inject into the mice?

A
  • Mec1 cells
  • transformed B cell leukemia cell
  • these cells grow by mesh/graphing with the other cells in the microenvironment
  • express CD19
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4
Q

What where the two major treatments in this experiment?

A
  • Clodrolip

- alphaCSF1R

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5
Q

When is CD20 expressed on a B-cell?

A

Expressed on B cells in the pro-B cell stage and increases in expression as the cell matures
- previously expressed CD19

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6
Q

What do monocytes differentiate into?

A

Macrophages

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7
Q

Where does the most DNA damage occur in normal metabolism?

A

DNA damage occurs in mtDNA due to the low amounts of DNA repair enzymes

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8
Q

Why did they use immunocompromised mice?

A
  • It gave the ability for the MEC1 cells to graft
  • could insert more human like cells
  • the experiment would have been more difficult in WT cells
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9
Q

What is CLL?

A

The prototype of chronic B cell tumors

- an adult neoplasia of B cells

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10
Q

What does the development and progression of CLL depend on?

A

Depends on a complex network of cells including macrophages

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11
Q

What did the paper describe?

A

It described a set of molecular interactions supporting the in vivo dependence of leukemic cells on monocytes/macrophages

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12
Q

What did the paper suggest?

A

It suggested therapeutic strategies based on macrophage targeting

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13
Q

What type of mice did they use in the investigation?

A

The Rag2-/- gammac-/- compound mutant mice

  • double knock out
    aka. Rag2/II2rg
  • exhibit T cell, B cell and NK cell immunodeficiencies that make them effective transplant hosts for human immune cells
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14
Q

What cell transplants are used to create a humanized model?

A
  • Hematopoietic stem cell
  • progenitor cell
  • can be used to develop and test novel immune therapeutic strategies
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15
Q

What two things were knocked out in the mice?

A
  • recombination activating gene 2 (Rag 2)

- Interleukin 2 receptor, gamma chain

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16
Q

What are RAGs?

A
  • genes that encode enzymes that play an important role in the differentiation in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules
  • expression is restricted to lymphocytes during their developmental stages
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17
Q

Why are RAGs essential?

A

They’re essential to the generation of mature B and T lymphocytes (components of the adaptive immune system)
- need a robust amount to have a varied amount of receptors to recognize various antigens

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18
Q

Why was the IL-2 receptor gamma chain knocked out?

A

When a GF binds, the IL-2 receptor gamma chain complexes with another tyrosine kinase called JAK3 which allows for growth, survival, transcriptional regulation or/and effector differentiation to occur
- when knocked out, cells lose their ability to grow or respond to IL-2

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19
Q

How is the IL-2 receptor activated?

A
  • trimerizes
  • not autophosphorylated
  • adapter molecules (JAKs) come in (near membrane) and bind to the receptor and function as a kinase
  • JAKs phosphorylate the beta subunit
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20
Q

What is the primary role of CD19?

A

It acts as a B cell as a coreceptor in conjuntion with others

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21
Q

What occurs to CD19 when the B cell is activated?

A

The cytoplasmic tail of CD19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3

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22
Q

What are myeloid-derived suppressor cells (MDSC)?

A
  • They are a heterogenous group of immune cells from the myeloid lineage
  • they strongly expand in pathological situations such as chronic infections and cancer
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23
Q

What do MDSCs do?

A
  • they interact with other immune cell types including T cells, dendritic cells, macrophages and natural killer cells to regulate their functions
  • possess strong immunosuppresive activities
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24
Q

What is CD20?

A

An activated-glycosylated phosphoprotein expressed on the surface of all B cells beginning at the pro-B phase increasing in the concentration until maturity

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25
Q

What is Ly6C?

A

A marker for monocytes in bone marrow also identifies MDSCs and activated macrophages in inflammatory tissues
- expressed by monocytic MDSCs

26
Q

What is CD11b?

A
  • integrin alphaM
  • is one subunit that forms the heterodimeric integrin alphaM-beta2 molecule, also known as macrophage-1 antigen (Mac-1) or complement receptor 3 (CR3)
27
Q

What is the role of CD11b?

A

It mediates inflammation by regulating leukocyte adhesion and migration and has been implicated in several immune processes
- such as phagocytosis, cell-mediated cytotoxicity, chemotaxis and cellular activation

28
Q

What is F4/80?

A

EGF like GPCR adhesion molecule

29
Q

What is MRC1?

A

Mannose receptor 1 (macrophage), endocytic receptor

30
Q

What is TNF-alpha?

A
  • Tumor necrosis factor-alpha pathway

- induces apoptosis

31
Q

What is TRAIL?

A
  • TNF-related apoptosis-inducing ligand
  • a ligand that binds to death receptor 5 (DR5) that induces apoptosis
  • its’s a member of the tumor necrosis factor receptor superfamily
32
Q

In Figure 1, picture B, why was there no uninfused cells?

A
  • Just looking at MEC1 cells
  • overt leukemia had a significant increase of % CD19 from the early stage
  • showed proof of concept
33
Q

In Figure 1, picture C, why were they looking at CD11b and CSF1R?

A

To see the effects of the MEC1 cells on the monocytes as they become macrophages

34
Q

What cells did they look at in Figure 1?

A
  • They looked at the cells in the bone marrow

- Gated on all CD45+ cells (all leukocytes - no erythrocytes)

35
Q

What were the results for monocytes (CSF1R) and TAMs (F4/80, MRC1, CD86 AND CSF1R)?

A
  • gave the characteristics of TAMs
  • monocytes: CSF1R more early, less later
  • TAMs: F4/80 less later
  • TAMs: more MRC1, CD86, CSF1R
36
Q

What transcriptional changes occurred in the MEC1 cells?

A

TAMs:
- increased TNF
CLL:
- increased CCL2 (a potent monocyte chemoattractant)
- decreased PTEN
- increased IL-10 (anti-inflammatory and suppresive)

37
Q

What was particularly relevant about the transcriptional changes in leukemic MEC1 cells?

A

The differential expression of genes supporting the existence of monocyte/macrophage-leukemic cell cross talk
- includes IL10 and CCL2

38
Q

What is F4/80?

A
  • a member of the adhesion GPCRs receptors

- required for the induction of efferent CD8+ regulatory T cells

39
Q

What happened to the spleen when it was treated with clodrolip (lipidsome encapsulated bisphosphonate)?

A
  • clodrolip targets osteoplasts

- the spleen reduces in size due to the clodrolip

40
Q

What were the effects of clodrolip in the bone marrow, spleen, PB and PE?

A

The % of hCD19 decreased once treated

41
Q

What is CD5?

A
  • found on a subset of IgM-secreting B cells called B-1

- also expressed in chronic lymphocytic leukemia

42
Q

How were the levels of F4/80 and other double positive cells effected by clodrolip?

A

The levels dropped

43
Q

How are IV and SC different in this experiment?

A

IV - injected directly into the bloodstream
SC - injected into the MEC1 cells directly
- though both were found to have similar effects

44
Q

What happened to the size of the lymph nodes and tumor when the clodrolip was injected directly into them?

A

The volume of the locations decreased after 48 days

- increased the lifespan of the treated mouse by 20 days

45
Q

What does clodrolip effect?

A
  • there is no direct effect on the tumor cells

- clodrolip has an affect on the TAMs in the microenvironment

46
Q

What is the apoptotic phenomenon?

A

Non-apoptotic cells: phosphatidylserine (PS) is inside of the membrane
Apoptotic cells: phosphatidylserine (PS) is outside of the membrane
- signals to the neighbouring cells that the cell is dying
- phosphatidylserine (PS) is recognized by annexin 5

47
Q

What is PI+ used for?

A
  • Can be used to identify which cells are necrotic

- enters the membrane through broken areas and interpolates with DNA -> fluoresence

48
Q

What information does annexin V+/Pi+ provide?

A

It can give you a ratio of cells that are apoptotic or necrotic

49
Q

What is clodrolip-mediated macrophage killing associated with?

A

It is associated with a drastic reduction of CLL growth in two mouse models of the disease

50
Q

What occured to the absolute numbers of the CD19, etc when treated with alphaCSF1R blockade?

A

The absolute number for all of the surface molecules decrease
- targets the monocyte/macrophage population

51
Q

When treated with alphaCSF1R and alphaCD20, how was the lifespan of the mouse effected?

A

The number of live animals had doubled at the end of 44 days

- extends the day of death

52
Q

What did clodrolip and anti-human CSF1R mAb effect?

A
  • Had no direct toxicty on leukemic B cells in vitro

- effect the monocytes/macrophages

53
Q

Why did levels of hCD19+ increase when treated with clodrolip and etanercept or alphaCSF1R and etanercept?

A
  • etanercept is a TNF inhibitor
  • would prevent the TNFs, released from the dying TAMs, from binding to the TNF receptors on the leukemic cells
  • can enhance the survival of CLL cells
54
Q

What are hCD68 and Ki67?

A

hCD68: expressed on monocytes/macrophages
Ki67: expressed on proliferating B cells
- together they show the proximity between the two types of cells and the importance of that

55
Q

What is PBMC?

A
  • peripheral blood mononuclear cells
  • heterogenous population of cells
  • contains: B cells, T cells and monocytes
56
Q

What was PBMC used for?

A
  • perfomed the experiment in vitro (animal was in vivo)

- negative purification to isolate B cells

57
Q

What occurred to the CD19/CD5 % cell depletion when PBMC, PMBC with separated monocytes and PBMC without monocytes were treated with clodrolip?

A
  • PBMC: there was 40% cell depletion
  • PMBC with separated monocytes: there was only a 20% cell depletion
  • PBMC without monocytes: there was only a 10% cell depletion
58
Q

Where does CLL occur?

A
  • In a permissive tissue microenvironment

- different cell types influence the disease progression and provide a basis for designing novel treatments

59
Q

Which certain intervention could aid in future therapy?

A

Malignant lymphocytes depend on interactive support from TAMs -> breaking this

60
Q

Why was bone marrow so important in the experiment?

A

It was found that this location was a critical niche for the leukemic MEC1 cells to engraft and progress with the help of monocytes/macrophages

Biology 465 (15 decks)

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