Dendritic Cells

Question 1

What are the three subsets of DCs?

Answer 1

Convention, plasmacytoid and innflamatory

Question 2

What are properties of Convention DCs?

Answer 2

Located at body surfaces and secondary Lymphoid tissues
Bring pathogenic material to draining LNs
Activate Naive T-cell responce

Question 3

What are properties of palsmacytoid DCs?

Answer 3

Circulate in blood periphery
Recruited to inflammation sites
Have immunomodulatory role
Produce Type 1 Interferon

Question 4

What are properties of Inflammatory DCs

Answer 4

Not found in steady state, only during Inflammation
Monocyte Derived
Increase antigen presentation during inflammation
Re-activate Memory T-cells in non-lymphoid tissues

Question 5

Markers for cDC1s human and mouse

Role of cDC1

Answer 5

Human: CD141, XCR1, CLEC9A, CADM1.
Mouse: CD103/CD8, XCR1, CLEC9A, CADM1

Role: Cross presentation, production of IL-12 + INF gamma, TLR3 expression, Th1/Th2 generation

Question 6

Markers for cDC2s in humans and mouse

Role of cDC2

Answer 6

Human: CD1c, CD11b, SIRP alpha
Mouse: CD24, CD11B, SIRP alpha

Role: DC4 T- Cell responce, IL-1B, IL-6, IL-23 production,
Expression of all TLR except TLR3 in mice and TLR9 in humans, Th2 and Th17 generation

Question 7

Where are Conventional and Plasmatoid DCs derived from?

Answer 7

Common Dendritic Progenitors

Question 8

Where are Inflammatory DCs derived from?

Answer 8

Monocytes

Question 9

What is the Process of a DC immune responce?

Answer 9

DCs in pheripheral tissues pick up antigens and express them on MHCs
DCs then become mature and migrate to Draining lymph nodes via the lymphatic vessels
Present antigens to T-cells in lymph nodes

Question 10

Whats the differences between mature and immature DCs?

Answer 10

Immature - high phagocytosis, Low MHC expression and low co-stimulatory molecules, Low CCR7
Mature: Low phagocytosis, high MHC expression and co-stimulatory molecules and high CCR7

Question 11

What causes DC maturation?

Answer 11

PAMPS:
Bacterial LPS (detected by TLR4) viral dsRNA (TLR3), Cytokines (TNF alpha and IL-1) and CD40 ligands

Question 12

How can you generate DCs from peripheral blood?

Answer 12

Isolate CD14 monocytes,
Subject them to IL-4 and GM-CSF (6days) to get an immature DC
Subject them to PAMPs, TNFalpha, IL-1beta 24 hours to get mature DC

Question 13

What is the fate of naive Th Cells based on the three signals 1,2 and 3?

Answer 13

Signal 1: MHC II expression of antigen to T-cell receptor leads to T-cell activation
Signal 2: CD80/CD26 on APC interact with T-cell CD28, this promotes T-Cell survival, IL-2 production and clonal expansion
Signal 3: APC secrete cytokines, this polarieses T-Cells

Question 14

What cytokine signals produce which Th Cell responce?

What Do these Th cell responses target?

Answer 14

IFNgamma - Th1 - Intracellular pathogens, virusus, mycobacterium, activation of macrophages
IL-4- Th2- Extracellular pathogens, helminths,Aid B-cells
1l-17-Th17-Bacteria/fungi (surface tissues recruit neutrophils).

Question 15

What type of T-Cell responce can DCs trigger in cancer and autoimmune immunotherapy?

Answer 15

Effector T-cells - cancer

Tregs - Autoimmuntiy

Question 16

How can DCs be isolated?

Answer 16

cd14+ monocytesm, DC generation, DC antigen loading,
Antigen presentation to T-cells
Maturation state of DCs determines T-Cell response

Question 17

Differecne between immature, semi mature and mature DCs in terms of MHC II, CD80/86, IL-12/IL-6 and TGF beta

Answer 17

Immature: MHC II + , CD80/86 - , IL-12/IL-6 - , TGFbeta -
Semi- Mature: MCH II + + , CD80/86 + , IL-12/IL-6 - , TGFbeta ++
Mature : MHC II ++, CD80/86 ++, IL12-IL-6 ++ , TGFbeta +

Question 18

Why use DCs for cancer therapy?

Answer 18

DC have ability to remove rare events
Other surgeries/ radio and chemotherapy dont always remove residual cancer cells which can re-occur. DC can clean up these residual cancer cells.
DCs non-toxic and target heterogenous tumors
can have multiple antigen targets and epitope spreading

Question 19

Negative of DCs for cancer therpay

Answer 19

Not good at removing bulky tumors

Question 20

Goals of DC based cancer therapy

Answer 20

1) Cancer specifc T-cell expansion
2) T-cells with appropriate Effector function
3) T-cells migrate to relevent tumor tissues

Question 21

Problems of DC cancer therapy?

Answer 21

No pro-innflamtory signals in cancers (eg no TLR ligands)
Tumors produce suppressive factors ie IL-10 TGFbeta which can lead to DC dyfunction
Cancer patients - expansion and hyperactivation of Tregs

Question 22

Desired DC features for cancer therapy

Answer 22

High MHC II/Co-stimulatory factors
High IL-12 to induce CTL and Th1
High CCR7 so DC migrate to lymph nodes
Attraction of desired immune cells - NK cells, CTL and CTH but avoid Tregs by appropriate chemokine profile eg high CCL5,CXCL10,CCL22
Resistant to supression - Final maturation

Question 23

3rd Generation DC cancer therapy

Answer 23

1st decribed by Radomski et al.,
CD14 monocyte isoloation, treat with IL-4 and GM-CSF, Which produces immature DCs. Treat with TNF alpha IL-1 INFgamma/alpha and Poly I:C to stimulate TLR3
Called it alphaDC1

Question 24

What was AlphaDC1s profile?

What are proplems with 3rd Gen DC cancer therpay?

Answer 24

MHC II +++, CD80/86+++ , CCR7+++, IL-12+++, CCL5/CXXL10 +++

Problems: Monocyte derived, so dont migrate well to lymph nodes (aprox 4%)

Question 25

4th Gen DC cancer therapy | Problems with it

Answer 25

Same as 3rd gen but used blood plasmacytoid or conventional DCs. This means no culture period needed and better migratory power to LN1 (1st clinical trail improved progression free survival in melanoma patients) Problems: Semi maturen DCs unstable and can make CD4 T-cells tolerogenic and inducy CD8 T-cell immunity.

Question 26

What are the properties of tolerogenic DCs?

Answer 26

MHC II ++ , CD80/86 + , TNFalpha / IL-12 - , TGFbeta/IL-10 ++

Question 27

How can you stabilise tolDCs

Answer 27

CD14 monocytes + IL-4/GM-CSF You then get immature DCs Culture with IL-10 and TGF beta Then can use Drugs such as Dex, Vit3D AND BAY11-7082

Question 28

What has TolDC induction shown in animal models?

Answer 28

Delay of Transplantation rejection and or prevntion of autoimmue diseases (autoimmune diseases can be cured)

Question 29

Work At Newcastle with tolDCs to treat RA purpose

Answer 29

re-establish tolerogenic responce against self antigen without affecting protective immunity

Question 30

Pros and conc of TolDCs for RA treatment

Answer 30

Pros: Low toxcicity, Targeted immunosuppression, Potential to restore underlying defect. Cons: Custimised and therefor high COST, Autoantigens still not known, Risk of enhancing autoimmune responce

Question 31

What they wanted to achive with tolDC treatment forn RA

Answer 31

Presentation of self autoantigen (high signal 1) Low co-stimulation, but this maybe needs to be changed to high (as co stimulation signal 2 needed for Treg induction) Anti-innflammatory cytokine profile (signal 3)

Question 32

How they achived tolDCs

Answer 32

Day 0 - CD14 monocyte isolation and culture with IL-4 and GM-CSF Day 3 - Culture of DCs with Dex, IL-4 and GM-CSF to generate immature DCs Day 6 - Immature DC cultured with Dex, Vit3D and TLR-4 ligand Day 7 - Mature TolDC

Question 33

What was the phenotype and cytokine profile of the TolDCs used for the RA treatment at newcastle?

Answer 33

Phenotype : MHC II +++, CD80/86 ++, CD83+/- Cytokine Profile: IL-10+++, TGFbeta +++ , IL-12 - , IL-23/IL-6/TNF + These DCs very good at polarising T-cells to inflammatory responce phenotype in mice. Mice showed reduced IL-17 and Very high IL-10

Question 34

AuTo DECRA

Answer 34

Phase 1 clinical trial. Unblinded, placebo controlled dose escalation trail to assess safety, tolerability, feasibility and efficacy of biomarkers. Conclusion - Safe (no knee flares) TolDCs local not systemic.