Targeting Macrophages Sensitizes Chronic Lymphocytic Leukemia to Apoptosis and Inhibits Disease Progression Flashcards
In this paper, where was the experiment taking place?
- In the mice bone marrow microenvironment
- in vitro culture samples from humans
What did the addition of clodrolip result in?
- 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
What type of leukemic cell did they inject into the mice?
- Mec1 cells
- transformed B cell leukemia cell
- these cells grow by mesh/graphing with the other cells in the microenvironment
- express CD19
What where the two major treatments in this experiment?
- Clodrolip
- alphaCSF1R
When is CD20 expressed on a B-cell?
Expressed on B cells in the pro-B cell stage and increases in expression as the cell matures
- previously expressed CD19
What do monocytes differentiate into?
Macrophages
Where does the most DNA damage occur in normal metabolism?
DNA damage occurs in mtDNA due to the low amounts of DNA repair enzymes
Why did they use immunocompromised mice?
- 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
What is CLL?
The prototype of chronic B cell tumors
- an adult neoplasia of B cells
What does the development and progression of CLL depend on?
Depends on a complex network of cells including macrophages
What did the paper describe?
It described a set of molecular interactions supporting the in vivo dependence of leukemic cells on monocytes/macrophages
What did the paper suggest?
It suggested therapeutic strategies based on macrophage targeting
What type of mice did they use in the investigation?
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
What cell transplants are used to create a humanized model?
- Hematopoietic stem cell
- progenitor cell
- can be used to develop and test novel immune therapeutic strategies
What two things were knocked out in the mice?
- recombination activating gene 2 (Rag 2)
- Interleukin 2 receptor, gamma chain
What are RAGs?
- 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
Why are RAGs essential?
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
Why was the IL-2 receptor gamma chain knocked out?
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
How is the IL-2 receptor activated?
- 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
What is the primary role of CD19?
It acts as a B cell as a coreceptor in conjuntion with others
What occurs to CD19 when the B cell is activated?
The cytoplasmic tail of CD19 becomes phosphorylated, which leads to binding by Src-family kinases and recruitment of PI-3
What are myeloid-derived suppressor cells (MDSC)?
- They are a heterogenous group of immune cells from the myeloid lineage
- they strongly expand in pathological situations such as chronic infections and cancer
What do MDSCs do?
- 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
What is CD20?
An activated-glycosylated phosphoprotein expressed on the surface of all B cells beginning at the pro-B phase increasing in the concentration until maturity
What is Ly6C?
A marker for monocytes in bone marrow also identifies MDSCs and activated macrophages in inflammatory tissues
- expressed by monocytic MDSCs
What is CD11b?
- 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)
What is the role of CD11b?
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
What is F4/80?
EGF like GPCR adhesion molecule
What is MRC1?
Mannose receptor 1 (macrophage), endocytic receptor
What is TNF-alpha?
- Tumor necrosis factor-alpha pathway
- induces apoptosis
What is TRAIL?
- 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
In Figure 1, picture B, why was there no uninfused cells?
- Just looking at MEC1 cells
- overt leukemia had a significant increase of % CD19 from the early stage
- showed proof of concept
In Figure 1, picture C, why were they looking at CD11b and CSF1R?
To see the effects of the MEC1 cells on the monocytes as they become macrophages
What cells did they look at in Figure 1?
- They looked at the cells in the bone marrow
- Gated on all CD45+ cells (all leukocytes - no erythrocytes)
What were the results for monocytes (CSF1R) and TAMs (F4/80, MRC1, CD86 AND CSF1R)?
- gave the characteristics of TAMs
- monocytes: CSF1R more early, less later
- TAMs: F4/80 less later
- TAMs: more MRC1, CD86, CSF1R
What transcriptional changes occurred in the MEC1 cells?
TAMs:
- increased TNF
CLL:
- increased CCL2 (a potent monocyte chemoattractant)
- decreased PTEN
- increased IL-10 (anti-inflammatory and suppresive)
What was particularly relevant about the transcriptional changes in leukemic MEC1 cells?
The differential expression of genes supporting the existence of monocyte/macrophage-leukemic cell cross talk
- includes IL10 and CCL2
What is F4/80?
- a member of the adhesion GPCRs receptors
- required for the induction of efferent CD8+ regulatory T cells
What happened to the spleen when it was treated with clodrolip (lipidsome encapsulated bisphosphonate)?
- clodrolip targets osteoplasts
- the spleen reduces in size due to the clodrolip
What were the effects of clodrolip in the bone marrow, spleen, PB and PE?
The % of hCD19 decreased once treated
What is CD5?
- found on a subset of IgM-secreting B cells called B-1
- also expressed in chronic lymphocytic leukemia
How were the levels of F4/80 and other double positive cells effected by clodrolip?
The levels dropped
How are IV and SC different in this experiment?
IV - injected directly into the bloodstream
SC - injected into the MEC1 cells directly
- though both were found to have similar effects
What happened to the size of the lymph nodes and tumor when the clodrolip was injected directly into them?
The volume of the locations decreased after 48 days
- increased the lifespan of the treated mouse by 20 days
What does clodrolip effect?
- there is no direct effect on the tumor cells
- clodrolip has an affect on the TAMs in the microenvironment
What is the apoptotic phenomenon?
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
What is PI+ used for?
- Can be used to identify which cells are necrotic
- enters the membrane through broken areas and interpolates with DNA -> fluoresence
What information does annexin V+/Pi+ provide?
It can give you a ratio of cells that are apoptotic or necrotic
What is clodrolip-mediated macrophage killing associated with?
It is associated with a drastic reduction of CLL growth in two mouse models of the disease
What occured to the absolute numbers of the CD19, etc when treated with alphaCSF1R blockade?
The absolute number for all of the surface molecules decrease
- targets the monocyte/macrophage population
When treated with alphaCSF1R and alphaCD20, how was the lifespan of the mouse effected?
The number of live animals had doubled at the end of 44 days
- extends the day of death
What did clodrolip and anti-human CSF1R mAb effect?
- Had no direct toxicty on leukemic B cells in vitro
- effect the monocytes/macrophages
Why did levels of hCD19+ increase when treated with clodrolip and etanercept or alphaCSF1R and etanercept?
- 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
What are hCD68 and Ki67?
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
What is PBMC?
- peripheral blood mononuclear cells
- heterogenous population of cells
- contains: B cells, T cells and monocytes
What was PBMC used for?
- perfomed the experiment in vitro (animal was in vivo)
- negative purification to isolate B cells
What occurred to the CD19/CD5 % cell depletion when PBMC, PMBC with separated monocytes and PBMC without monocytes were treated with clodrolip?
- 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
Where does CLL occur?
- In a permissive tissue microenvironment
- different cell types influence the disease progression and provide a basis for designing novel treatments
Which certain intervention could aid in future therapy?
Malignant lymphocytes depend on interactive support from TAMs -> breaking this
Why was bone marrow so important in the experiment?
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
